1. Preparation of Samples
<ol>
	<li>Dilute the cell suspension with ORFLO diluent or an appropriate diluent so that the cell concentration is within the operating range of the instrument (3,000 to 500,000 cells/mL for Type M cassette, or 3,000 to 2,500,000 cells/mL for Type S cassette).</li>
	<li>A dilution of 1:5 to 1:20 (Type M cassette) or no dilution to 1:5 dilution (Type S cassette) is recommended for most mammalian cell lines, but the appropriate dilution will depend on cell type and seeding density. The volume required for an accurate count is approximately 75 &mu;L.</li>
</ol>
2. Running Samples
<ol>
	<li>Turn the Moxi Z on by pressing the power button and the Home screen will be displayed.</li>
	<li>Press the tray down and insert a cassette into the Moxi Z. The Pipette 75&mu;L Sample... screen will be displayed.</li>
	<li>Pipette a 75 &mu;L sample into the fill port of the cassette (blue oval labeled 1 or 2, depending on which end of the cassette was inserted into the instrument). These are disposable cassettes, which can be used for 2 tests. Note: Place the pipette tip directly into the loading well at ~45&deg; angle so that the tip is caught under the front lip of the well. When dispensing, it is important that the vacuum does not "get ahead" of the dispensing of the fluid as could cause pockets of air to enter the cassette. It is recommended to have a small bead (the size of entry well) of fluid form over the well during dispensing.</li>
	<li>For counting most mammalian cells, touch the screen anywhere to start. For counting very small particles (4 to 8 &mu;m average diameter for Type M and 3-8 &mu;m average diameter for Type S), the Moxi Z can be run in Small Particle Mode. Note: the cassette type is indicated in the black bar at the top of the screen. In this mode, Moxi Z sets the diameter scale to 2 to 10 &mu;m as the default and performs the count using optimized parameters for the detection of small cells. Press the TOUCH HERE Small Particle Mode button (black square) to initiate the test and run in this mode.</li>
	<li>The Moxi Z will begin the test and the cell count results will be complete in approximately 8 seconds (type M cassette) or 15 seconds (Type S cassette).</li>
	<li>The Curve Fitting and MVI calculations begin automatically and require only a few additional seconds. When a suitable fit is found, the results will then automatically be displayed on the screen.</li>
	<li>To make Gating the default acquisition mode, press the Curve Fit Count button to toggle into Gating mode.</li>
	<li>At this point the cassette can be removed from the unit.</li>
</ol>
3. Managing Data
<ol>
	<li>If AutoMode scaling is off, the results are initially displayed for a curve fit count on a diameter scale of 2-34 &mu;m (Type M cassette) or 2-26 &mu;m (Type S cassette). If Automode scaling is on, then the Moxi Z will automatically scale the horizontal axis (x-axis) to the range that is closest to the width of the curve-fit population while ensuring all curve-fit data is displayed.</li>
	<li>If AutoMode scaling is off, to manually display the results at a higher resolution, touch the 2-26 &mu;m icon (Type M) or the 2-18 &mu;m icon (Type S). Note: The cassette type is indicated in the blue box at the top left of the screen. This is recommended when counting smaller cells or particles to improve both the curve-fitting capabilities as well as the user's ability to visually discriminate nuances of the data.</li>
	<li>Upon increasing the horizontal resolution, the scaling button will dynamically adjust to allow for cycling between the available x-axis scale ranges: 2-34, 2-26, 2-18, and 2-10 &mu;m for the Type M cassette; or 2-26, 2-18, and 2-10 &mu;m for the Type S cassette. This feature is only available immediately following a cell count.</li>
	<li>Count information (cells/ml) for the curve-fit or gated region is displayed in a black box under the left side of the histogram. Mean cell size information is displayed in the black box under the right side of the histogram.</li>
	<li>The MVI value is displayed a blue box at the top right of the histogram. If the test was run in small particle mode, the MVI does not apply and will be displayed as "MVI=SPM". Additionally if the concentration of cells is outside of the MVI operating range, a message of "MVI=Out of Range" will be displayed.</li>
	<li>To save the current histogram, touch the Done icon. This saves the data with a name of "Test XXX" where XXX is the corresponding test number.</li>
	<li>To manually gate the histogram, touch to toggle the Curve Fit count results button. This makes Gating the default acquisition mode. Then slide each blue gating marker to set the gates or tap the left and right arrows (these appear upon touching the blue gating marker) for fine gate adjustments Only the cells between the markers are counted. The histogram vertical scale is automatically scaled based on the curve-fit population amplitude. To adjust this vertical scale, swipe the screen up or down in the histogram region.</li>
	<li>Touch the Gated Count results button to return the display to curve fit mode and make curve fit mode the default acquisition mode.</li>
	<li>Then press the Done icon to save the results and return to the Home screen. To delete the results, press the Delete icon at any time to permanently delete the results of the test.</li>
</ol>
4. Analysis of Previously Acquired Data
<ol>
	<li>To open a saved test, press the Histogram icon on the Home screen.</li>
	<li>Icons for up to nine saved histograms will be displayed on the screen. Press the appropriate icon for the test of interest or press the Page Up or Page Down icons to view more test results.</li>
	<li>Each histogram will be opened in either the Curve Fit Count mode or Gated Count mode, depending on how it was saved. In Gated Count mode, gating markers can be positioned as desired by sliding each blue gating marker independently. Auto Gate by touching on the desired peak. Touch to toggle between the Gated Count and Curve Fit Count modes.</li>
	<li>Press the Zoom and Unzoom icons to adjust the vertical scale of the histogram. The vertical scale can also be changed by vertically swiping a finger on the display up (to increase) or down (to decrease).</li>
	<li>Press the Delete icon to permanently delete the results of the test.</li>
	<li>Press the Done icon to close the test results and return to the Home screen. (Note: zoomed view will not be saved.)</li>
</ol>
5. Transferring Data to a PC
<ol>
	<li>Data can be transferred to a PC using the Orflo Moxichart application or via USB transfer by plugging the Moxi Z USB cable to a PC or Mac. For Bluetooth transfer (MoxiChart), first determine the unit's Bluetooth ID by pressing the Bluetooth icon on the Home screen of the Moxi Z unit.</li>
	<li>On the computer, open the MoxiChart application and click on Bluetooth icon in upper right corner. Then, choose the device which corresponds to the Bluetooth ID of the Moxi Z and select a destination folder for the uploaded files.</li>
	<li>Moxichart will automatically transfer all the files to the specified directory via the Bluetooth connection.</li>
	<li>Files transferred by Bluetooth (MoxiChart) or USB can be opened and analyzed directly with the MoxiChart application or, because they are formatted .csv files, they can be directly opened in Microsoft Excel or other data analysis programs for additionally/custom analysis.</li>
</ol>
6. Representative Results
To assess the precision and accuracy of the Moxi Z, counts of HEK-293 cells, HeLa cells, CHO-K1, Yeast, Jurkat E6-1 cells, PC12 cells, and precision-calibrated bead suspensions with concentrations ranging from 0-500,000 cells/mL (Type M cassette) and 0-2-2.5e6 cells/ml (Type S cassette) were counted and compared using a Coulter Z2 and the Moxi Z. All mammalian cells were obtained from the American Type Culture Collection (ATCC) and cultured as previously described7-11. Briefly, cells were cultured in suspension in 75 mm2 tissue culture flasks with core media of RPMI 1640 (Jurkat E6-1, PC12), MEM (HEK-293, HeLa) cells, or F12K(CHO-K1) cells. All media was supplemented with 10% fetal bovine serum and 100 U penicillin/100 &mu;g Streptomycin. Flasks were maintained in an incubator at 37 &deg;C with 5% CO2. Cells were passaged every 3 days. Yeast cells (X5, C. albicans, Vin 13) were donated and used immediately as provided. Initial sample concentrations of ~500,000 cells/ml for the Type M cassette data and ~2-2.5e6 cells/ml for the Type S cassette were established using the Coulter Z2. Subsequent theoretical concentration levels were prepared through serial dilutions with physiological buffers (Isoton II or Hank's Balanced Salt Solution). At each concentration, identical samples were measured by both systems and plotted against each other. As seen in Figure 2, there was a strong linear correlation (Type M r2&gt;0.9886, Type S r2&gt;0.9781) between the counts for all samples. Concentrations were also compared to expected/theoretical cell concentrations with both the Moxi Z and the Z2 (Figure 3) and again demonstrated strong linear correlations (Type M r2&gt;0.9758, Type S r2&gt;0.9774).
To assess precision, HEK-293 and HeLa cell counts were performed using the Coulter Z2, Moxi Z, and a hemocytometer. The mean coefficient of variation (CV, n=19) of cell counts using the Moxi Z (Type M cassette), Coulter Z2 and a hemocytometer were calculated with CV's measured from 5 separate counts in the 200,000-300,000 cells/ml range. The small average CV (Figure 4) of the Moxi Z is comparable to that of the Z2 and is representative of the high level of precision only attainable with Coulter-based counting technologies.
Sizing precision of the Moxi Z instrument was evaluated next through the measurement of purchased, precision calibrated polystyrene beads. Averaged particle size measurements (n=5) from Moxi Z were plotted against manufacturer reported sizes. As can be seen in Figure 5, the Moxi Z measurements consistently matched the manufacturer values with a high linear correlation (r2=0.9989).
Beyond size and count information, Moxi Z provides a reagent-free, general assessment of mammalian cell culture health with a reported Moxi Viability Index (MVI) value. This index is generated from a ratio of the number of cells in the population of interest with respect to the total particulate counts. To demonstrate the MVI measurement capabilities, MVI readings (Type M cassette) were compared to standard manual and flow cytometric live/dead measurement techniques. Populations of dead (&lt;10% viable) HEK-293 and HeLa cells were created through overnight incubations of the live cells in a nutrient-free, sodium fluoride containing diluent (Isoton II, Beckman Coulter) at 37 &deg;C. Controlled theoretical viability levels were prepared by ratiometrically mixing known concentrations of live and dead cells. Resulting solutions were analyzed for viability levels in duplicate with both hemocytometer counts and flow cytometer measurements. Hemocytometer measurements were made using a traditional 50/50 mixture of 0.1% Trypan Blue Solution and cells. Flow cytometry measurements were made using a Guava PCA cytometer (Merck) using the Viacount reagent and the manufacturer-specified protocol (Merck). As shown in Figure 6, the Pearson correlation coefficient values (r2) "of the linear fit of the MVI data to analogous hemocytometer viability percentages for" HEK cells and HeLa cells were r2=0.9937 and r2=0.9728, respectively. These results demonstrate that this approach provides culture health information across a broad range of cell viabilities that compare favorably to the live/dead measurements obtained using a flow cytometer or hemocytometer.
Tables and Figures (From Dittami et al., 2011)15:
<img alt="Figure 1" src="/files/ftp_upload/3842/3842fig1.jpg" /> 
Figure 1. Thin-film Coulter Counting: Electrical current is passed through the Cell Sensing Zone of the thin-film cassette. As cells flow single file through the CSZ, the momentary increases in voltage are measured by the Moxi Z.
<img alt="Figure 2" src="/files/ftp_upload/3842/3842fig2.jpg" /> 
Figure 2. Moxi Z counts are comparable to Coulter Z2 counts. Identical counts of cell and bead suspensions were counted using both a Coulter Z2 and the Moxi Z. Averaged count values (n=3-4) of the Moxi Z counts were plotted with respect to the Coulter corresponding Z2 counts and r2 values of the linear fit were determined. A) Type M cassette - concentration range of 0 - 500,000 cells/ml. B) Type S cassette - concentration range of 0 - 2-2.5e6/ml.Error Bars indicate &plusmn; 1 standard deviation.
<img alt="Figure 3" src="/files/ftp_upload/3842/3842fig3.jpg" /> 
Figure 3. Moxi Z concentration measurements vs theoretical cell concentrations. A) Type M cassette -Theoretical concentration values were prepared from serial dilutions of an initial 500,000 cells/ml sample. B) Type S cassette -Theoretical concentration values were prepared from serial dilutions of an initial 2 - 2.5e6 cells/ml sample. Error Bars indicate &plusmn; 1 standard deviation.
<img alt="Figure 4" src="/files/ftp_upload/3842/3842fig4.jpg" /> 
Figure 4. Coefficient of Variation for the Coulter Z2, Moxi Z (Type M cassette), and Hemocytometer. The mean coefficient of variation (CV, n=19) of HEK-293 and HeLa cell counts for each approach were calculated with CVs measured from 5 separate counts of HEK-293 and HeLa Cells in the 200,000- 300,000 cells/ml range. Error Bars indicate &plusmn; 1 standard deviation.
<img alt="Figure 5" src="/files/ftp_upload/3842/3842fig5.jpg" /> 
Figure 5. Precision of particle size measurements using Moxi Z- Moxi Z measured bead size (Type M cassette) vs. manufacturer reported bead size. Error Bars indicate &plusmn; 1 standard deviation.
<img alt="Figure 6" src="/files/ftp_upload/3842/3842fig6.jpg" /> 
Figure 6. Culture Health Assessment using MVI - Comparison of MVI measurements (Type M cassette) and Guava PCA Viacount viability versus visual, trypan-blue stained viability counts using a hemocytometer.

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	<li>Fernyhough, M. E., Helterline, D. L., Vierck, J. L., Hill, R. A., Dodson, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coulter+counter+use+in+the+enumeration+of+muscle+and+fat+stem+cells.">Coulter counter use in the enumeration of muscle and fat stem cells.</a> Methods. Cell. Sci. 25, 221-225 (2003).</li><li>Blades, A. N., Flavell, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+use+of+the+Coulter+model+D+electronic+cell+counter+in+clinical+haematology.">Observations on the use of the Coulter model D electronic cell counter in clinical haematology.</a> J. Clin. Pathol. 16, 158-163 (1963).</li><li>Morris, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+Size+Analysis+of+Beer+Solids+using+a+Coulter+Counter.">Particle Size Analysis of Beer Solids using a Coulter Counter.</a> J. Inst. Brew. 90, 162-166 (1984).</li><li>Marth, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+Somatic+Cell+Counting+Procedure.">Electronic Somatic Cell Counting Procedure.</a> Standard Methods for the Examination of Dairy Products. , 134-140 (1978).</li><li>Parks, L. R., Jurbergs, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Number+and+size+distribution+of+particles+in+cellulosic+solutions.">Number and size distribution of particles in cellulosic solutions.</a> J. Appl. Polym. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrically+evoking+and+electrochemically+resolving+quantal+release+on+a+microchip.">Electrically evoking and electrochemically resolving quantal release on a microchip.</a> Lab on a Chip. 10, 30-35 (2010).</li><li>Nahreini, P., Hanson, A., Prasad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+production+of+recombinant+antibody+fragments+in+HEK-293+cells+using+sodium+butyrate.">High-yield production of recombinant antibody fragments in HEK-293 cells using sodium butyrate.</a> BioTechiques. 34, 968-972 (2003).</li><li>Escuyer, V., Collier, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthrax+Protective+Antigen+Interacts+with+a+Specific+Receptor+on+the+surface+of+CHO-K1+Cells.">Anthrax Protective Antigen Interacts with a Specific Receptor on the surface of CHO-K1 Cells.</a> Infection and Immunity. 59, 3381-3386 (1991).</li><li>Isaacs, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Borrelia+bugdorferi+bind+to+epithelial+cell+proteoglycans.">Borrelia bugdorferi bind to epithelial cell proteoglycans.</a> J. Clin. Invest. 93, 809-819 (1994).</li><li>Saito, Y., Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+selenoprotein+P+as+a+selenium+supply+protein.">Characterization of selenoprotein P as a selenium supply protein.</a> Eur. J. Biochem. 269, 5746-5751 (2002).</li><li>Kataoka, S., Tsuruo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physician+Education:+Apoptosis.">Physician Education: Apoptosis.</a> Oncologist. 1, 399-401 (1996).</li><li>Liegler, T. J., Hyun, W., Yen, T. S., Stites, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+live,+apoptotic,+and+necrotic+human+peripheral+lymphocytes+by+single-laser+flow+cytometry.">Detection and quantification of live, apoptotic, and necrotic human peripheral lymphocytes by single-laser flow cytometry.</a> Clin. Diagn. Lab Immunol. 2, 369-376 (1995).</li><li>Sheridan, J. W., Bishop, C. J., Simmons, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+and+morphological+correlates+of+kinetic+change+and+death+in+a+starved+human+melanoma+cell+line.">Biophysical and morphological correlates of kinetic change and death in a starved human melanoma cell line.</a> J. Cell. Sci. 49, 119-137 (1981).</li><li>Dittami, G. M., Rabbitt, R. D., Ayliffe, H. 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Gregory M. Dittami and H. E. Ayliffe are employees of Orflo Technologies, the company that designed, manufactures and sells the Moxi Z. Richard D. Rabbitt provides part-time, paid consulting expertise to Orflo Technologies.
Production and Free Access to this article is sponsored by Orflo Technologies.

The underlying implementation the Coulter approach can be highly deterministic of the overall accuracy of the measurements. A critical area is the correction of the raw cell counts for "coincidence events" where two or more cells simultaneously pass through the aperture and are measured as a single electrical event. "Coincidence events" contribute to error which varies depending on a number of factors including cell size and degree of clustering (Davis et al 1967)6. As a result, the required coincidence correction for any given sample cannot be predicted, varying widely between cell/particle types and even between different cultures of identical cell types. Established theory, rooted in initial publications by Coulter, involves applying a logarithmic adjustment of the raw counts based on the number of events observed and an empirically-determined, particle-specific correction factor, z. While accurate counts can be properly achieved with this correction algorithm, it is limited in its practical applicability due to the large variations in z values between cell types and the corresponding difficulty in accurately predicting its value. Here, using the "true" cell count identified by the Moxi Z curve fitting in conjunction with the coincidence correction algorithm, the Moxi Z dynamically corrects for coincidence to achieve the true concentrations. When compared to results obtained using high-end Coulter Z2, the Moxi Z produced comparable counts across a concentration range of 0 - 500,000 cells/ml (Type M cassette) and 3,000 - 2-2.5e6 cells/ml (Type S cassette). Furthermore, the Moxi Z achieves this count accuracy with a similar low coefficient of variation in counts and a precision in particle sizing that is characteristic of the gold standard, Coulter-technique in cell counting.
	Moreover, the data presented here indicate that the Moxi Z can provide valuable information regarding the overall health of cell cultures. The Moxi Z leverages the curve fitting approach with a proprietary software algorithm to determine this culture health assessment. Morphological changes associated with cell death, such as blebbing, breaking apart, and volumetric distortions (Kataoka and Tsuruo 1996, Liegler et al 1995, Sheridan et al 1981)12-14, make it possible for the Moxi-Z to distinguish impedimetric differences between live populations and dead cell/debris populations. The MVI is generated by analyzing the culture/particle size distribution to identify the relative contributions of particle debris, shrunken necrotic cells, and the curve-fitted cell population of interest. The MVI value thereby reflects a population index or ratiometric measure of the monodisperse population counts with respect to the overall particle population profile. This value is then algorithmically-adjusted based population statistics and empirical observations. Because the MVI looks at parameters other than traditional staining approaches, it is not expected to mirror these techniques but instead provides a valuable alternative view into the health of a cell culture, particularly with respect to the debris and microbial contaminants.
	In summary, the Moxi-Z cell counter is an ultra small benchtop instrument that provides precise, reproducible assays of cell count, cell size, and cell health using the Coulter Principle and curve-fitting software algorithms combined with a patented thin-film sensor technology. With the Type M cassette it is capable of counting particles ranging from 4- 25 microns in diameter (dynamic range of 2 - 34 microns), in 8 seconds. With the Type S cassette it is capable of counting particles ranging from 3- 20 microns in diameter (dynamic range of 2 - 26 microns), in 15 seconds. Furthermore, the Moxi-Z performs health assessments without the use of reagents. It's much less labor intensive and subjective than manual counting. In comparison to standard Coulter counting, the Moxi is faster, significantly smaller, provides more information, requires considerably less maintenance, is easier to use, and is a fraction of the cost.

<table border="1" >
	<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>ORFLO Diluent</td>
 <td>ORFLO</td>
 <td>MXA006</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cassette Dispenser</td>
 <td>ORFLO</td>
 <td>&nbsp;</td>
 <td>Stores up to 25 cassettes for convenient dispensing</td>
 </tr>
 <tr>
 <td>USB Cable</td>
 <td>ORFLO</td>
 <td>&nbsp;</td>
 <td>Connects instrument to PC/Mac or power adapter</td>
 </tr>
 <tr>
 <td>Power Adapter (US and EU models only)</td>
 <td>ORFLO</td>
 <td>&nbsp;</td>
 <td>Connects USB cable to an AC outlet</td>
 </tr>
 <tr>
 <td>USB Flash Drive</td>
 <td>ORFLO</td>
 <td>&nbsp;</td>
 <td>Stores Moxi Z software and user manual</td>
 </tr>
 <tr>
 <td>Calibration Check Beads</td>
 <td>ORFLO</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Electronic Calibration Cassette</td>
 <td>ORFLO</td>
 <td>&nbsp;</td>
 <td>Electronic cassette for verifying proper system operation and calibration</td>
 </tr>
 </tbody>
</table>

determination of mammalian cell counts, cell size and cell health using the moxi z mini automated cell counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

Watch this Scientific Journal Video about Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter at JoVE.com

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

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JoVE Journal

Biology

24.6K ビュー.  Orflo Technologies. Moxi Zミニチュア自動セルカウンターは、特許取得済みの薄膜センサー技術とサイジングを実行するために、独自のソフトウェアアルゴリズムを使用して、粒子の幅広いサイズの範囲のカウントだけでなく、単分散の全体的な健全性を判断するコールター原理を組み合わせた斬新な楽器です哺乳動物細胞培養。このプロトコルは培養細胞の健康をカウント?...

ビデオ: Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&amp;#945; Promoter Using Gibson Assembly

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...