Drs. C. J. P. Grimmelikhuijzen and Michael Williamson from University of Copenhagen (Denmark) are appreciated for providing the aequorin plasmid. Our collaborator, Dr. Ronald J. Nachman from ARS-USDA (TX, USA), is acknowledged for peptide synthesis and for providing the NOVOstar plate reader.

1. Establishment of stable cell lines
<ol>
	<li>Clone your GPCR of interest and insert it into an expression vector incorporating a 5' Kozak consensus sequence (GCCA/GCCATGG) around the start codon for optimal ribosomal binding in a mammalian system (Kozak, 1986). Here we use the plasmid pcDNA3.1/Bm-KR for the tick kinin receptor, and the plasmid pcDNA3.1/Aedae-KR for the mosquito kinin receptor (Holmes et al., 2003; Pietrantonio et al., 2005). The pcDNA3.1(-) vector encodes ampicillin resistance for selection in bacteria and neomycin (G418) resistance for selection in mammalian cells. </li>
 <li>Grow CHO-K1 empty cells (without plasmids) (ATCC, Manassas, VA, USA) or other desired cell line in a T-25 flask (BD Falcon) at 37&deg;C in a 5% CO2 humidified incubator (Holmes et al., 2003). All further incubations should be done under these conditions unless specified otherwise. Maintain the empty cells in growth medium (F12K medium with 10% fetal bovine serum) with 1X Antibiotic-Antimycotic (Invitrogen,CA). </li>
 <li>To split cells, warm up all solutions to 37 &deg;C. Remove old medium in the T-25 flask and rinse with 5 ml PBS and then remove PBS. To trypsinize cells, add 2 ml PBS-Trypsin-EDTA (34 ml PBS, 2 ml 7.5% sodium bicarbonate, 4 ml 10X trypsin-EDTA) for 2 min. Add 3 ml medium and aspirate medium up and down to mix cells. Transfer medium with cells into a conical tube and centrifuge for 2 min at &tilde;200-300 g (1,000 rpm). Discard supernatant and re-suspend cells in 5 ml medium. Dilute the resuspended cells to a concentration of 1:5 or 1:10 with fresh medium and transfer 5 ml in a new T-25 flask. </li>
 <li>After the cells are growing healthily (around 2-3 days), seed the CHO-K1 cells into T-25 tissue culture flasks and grow them overnight in growth medium without antibiotics until they are about 30% confluent (approximately 18 hours). The degree of confluency can be determined by observing cells under inverted fluorescent microscopy. </li>
 <li>Prepare the following mixtures for each sample:
 <ol>
 <li>Combine 1-2 &mu;g DNA (example: use 4&mu;l of 265&mu;g/&mu;l pcDNA3.1/Aedae-KR) with 100 &mu;l Opti-MEM I Reduced Serum Medium (Invitrogen).</li>
 <li>Mix 6 &mu;l Lipofectin Reagent (InvitrogenTM) into 100 &mu;l F12K serum free medium, making a 1:1 ratio of Lipofectin to DNA. Incubate at room temperature for 30-45 min.</li>
 </ol>
Gently mix the solutions from 1.5.1 with 1.5.2 in a drop-wise fashion (total volume = 200 &mu;L). Let stand at room temperature for 10-15 min.
</li>
 <li>Remove the old growth medium from the cells and wash the cells with 5 ml F12K serum free medium and then remove the F12K serum free medium. In a 15 ml tube, gently mix the transfection mixture into 1.8 ml of fresh F12K medium in a drop-wise fashion. Then, washed the cells from step 1.5 with PBS and add this new transfection solution to the cells. Incubate for 18 hours.</li>
 <li>Change the medium to F12K medium plus 10% fetal bovine serum without antibiotics and incubate overnight. Split the cells into two T-25 flasks with F12K medium plus 10% fetal bovine serum without antibiotics for another 18 hours (for splitting cells please see step 1.3).</li>
 <li>Replace the medium with selective medium (F12K medium plus 10% fetal bovine serum with 800&mu;g/ml GENETICIN, Invitrogen). Culture the cells using the selective medium for 3-4 weeks. Over time this will select for cells that have stably incorporated the plasmid into their genomic DNA. Continue maintaining the cells using maintenance medium (F12K medium plus 10% fetal bovine serum with 400&mu;g/ml GENETICIN). Periodically freeze cell lines with 1:1 ratio of freezing media (selective medium with 20% DMSO) to prevent loses in case of unexpected contamination.</li>
 <li>Selecting clonal cell lines: as a first step, trypsinize and centrifuge the cells from step 1.8 with maintenance medium as in step 1.3. Re-suspend cells in 5 ml maintenance medium, take 0.5 ml of the cell suspend and add 4.5 ml of fresh maintenance medium to make a 10x dilution. Transfer the 10x dilution into 12 wells of a 96 well plate, adding 100 &mu;l to each well to select for single cells.</li>
 <li>Continue to make 10x serial dilutions of these cells for a theoretical final suspension of one cell per 100 &mu;l (normally the final dilution will be in the range of 10-11 to 10-19; the total number of 10x serial dilutions is &tilde;19). Immediately following each dilution, transfer 100 &mu;l of the dilution into 12 wells of the 96 well plate. After 18 hours incubation, observe 96 well plates under an inverted light or fluorescence microscope and mark wells that appear to only contain one single cell or contain two cells that obviously divided from one single cell. Keep observing every day and change medium every three days.</li>
 <li>When the wells are 80% confluent (about a week), rinse marked wells with 200 &mu;l PBS and trypsinize them with 100 &mu;l of PBS-trypsin-EDTA solution. Transfer cells from each marked well into one well of a 6-well plate with 1 ml maintenance medium. Grow those cells for 3 days then transfer cells into individual T25 flask. Test these cells using the calcium bioluminescence assay with agonist peptides (please see section 2).</li>
 <li>Select 1 cell line from step 1.11 with the highest response in the calcium bioluminescence plate assay and perform for a second time the single cell selection following steps 1.9-1.11. Periodically freeze cell lines.</li>
 <li>From the secondary selection described in step 1.12, choose 2-3 cell lines with the highest response in the calcium bioluminescence plate assay and maintain them in culture for further calcium bioluminescence plate assays. Keep track of passage numbers. From time to time, freeze cell lines from early passages so that you can always go back to them if the cell lines with more passages stop performing consistently. </li>
</ol>
2. The calcium bioluminescence plate assay
<ol>
	<li>Ligate the reporter gene of interest into an expression vector. Here we used aequorin plasmid mtAEQ/pcDNA1 (a gift from Drs. C. J. P. Grimmelikhuijzen and Michael Williamson, University of Copenhagen, Denmark). Transform the plasmid in E. coli cells MC1061/PS (Invitrogen) and purify them using a QIAprep spin miniprep kit (Qiagen Inc.). In the final step elute the plasmid with Tris buffer without EDTA, not water.</li>
 <li>Grow cell lines from step 1.13 expressing the desired receptor in maintenance medium. When the cells are 90% confluent, trypsinize, centrifuge and then re-suspend the cells in 5 ml maintenance medium as in step 1.3. Dilute cells (about 10x with maintenance medium) and count cell number with cell counter (Bright-Line Hemacytometer) under microscopy. Adjust the cell number to approximately 2 x 105 cells/ ml (average 20 cells in one of the 9 squares showed in the Hemacytometer). Seed 2 ml diluted cells in media into each well of a six well plate. Incubate for 24 hours (the cells should reach about 60% confluency after incubation).</li>
<li>Change media in the 6 wells plate to OPTI-MEM medium. For each well, mix 96 &mu;l OPTI-MEM with 4 &mu;l FuGENE 6 Transfection reagent (Roche Biochemicals) in a microfuge tube and let sit at room temperature for 5 min. Add 1 &mu;g of aequorin/pcDNA 1 plasmid DNA into each tube and then gently shake the sample for 1 min, incubate at room temperature for 15-20 min. Add each mixture into each well in a dropwise fashion while gently shaking the well plate. Incubate the plates for 6 hours and change the medium to F12K medium containing 10% fetal bovine serum without antibiotic.</li>
<li>After incubating the cells in six well plate for 24 hours, trypsinize, centrifuge and re-suspend the cells as step 1.3. Count the cell number to 400,000 cells/ml as step 2.1 and transfer 100 &mu;l (total 40,000 cells/100 &mu;l) into each well of a 96-well white thin bottom microtitre plate (Costar 3610). Incubate for another 24 hours until about 80% cell confluency. This is the optimal concentration of cells for the bioluminescence assay.</li>
<li>Prepare 90&mu;l/well of a calcium-free DMEM media (Invitrogen) containing 5 &mu;M coelenterazine (Invitrogen) in the dark (coelenterazine is light sensitive). Take the plate from 2.4, remove old media and add this 90&mu;l into each well. Incubate plates for 3 hours in the dark at 37&deg;C and 5% CO2, after which the cells in the plate are ready to be tested.</li>
</ol>
3. Instrument operation and data analysis
<ol>
	<li>Each bioluminescence plate reader is different. We perform our assay using a NOVOstar (BMG Labtechnologies) plate reader in bioluminensence mode. If you use a different instrument, you must adapt the protocol.</li>
 <li>Purge the plate reader pumps (or PRIME PUMPS) before use. Turn off the light in the room before putting the plate on the plate holder. Once the plate holder has closed, turn the lights on. </li>
 <li>Solubilize peptides (in a 1.5 ml Eppendorf tube) in the calcium-free DMEM media. Set the "Aspirate Depth" and "Position Determination" of the peptide solution before use. Challenge the cells with 10 &mu;l (10x) of varying concentrations of the peptides (FFFSWG-NH2, Aedes-K1-3, or other desired peptide) and immediately begin recording the light emission. We have set the instrument to record the light emission (465 nm) for each well every 2 seconds for a total time of 50 seconds. </li>
 <li>Make sure to include a positive control such as an active analog (analog FFFSWGa has been used, Taneja-Bageshwar et al., 2009) and a negative control such as vector only transfected cells. The negative control will be necessary during data analysis to set the baseline threshold (see representative results). An unrelated, inactive peptide can also be added as a negative control. </li>
 <li>After the run is complete, wash the instrument pump (or PRIME PUMPS) then place your next peptide sample. Save your data and wash the pumps again. </li>
 <li>Data handling: Transfer the data of light emission from each well into a Microsoft Excel data sheet.</li>
 <li>Paste the data from Excel to Prism software 4.0 from GraphPad Software Inc. (San Diego, CA, USA). The various peptide concentrations is the X-axis and bioluminescence units is the Y-axis. To normalize the data, plot a log-response curve. Select a nonlinear regression curve fit analysis (sigmoidal dose-response equation with variable slope) to obtain concentration-response curves for each peptide. The program plots the values in the end and gives the EC50. </li>
 <li>Each experiment should be repeated three times for data analysis.</li>
</ol>
4. Representative Results: 
When expressed in CHO-K1 cells, the mosquito Aedes aegypti kinin receptor behaved as a multiligand receptor and functionally responded to concentrations as low as 1 nM of the three endogenours Aedes kinins, Aedae kinins 1-3, tested singly using the calcium bioluminescence plate assay. Figure 1 shows that the rank order of potency obtained was Aedae-K-3 &gt; Aedae-K-2 &gt; Aedae-K-1, based on the respective EC50 values of Aedae-K-3, 16.04 nM; Aedae-K-2, 26.6 nM and Aedae-K-1, 48.85 nM, which were statistically significantly different (P &lt; 0.05) (Pietrantonio et al., 2005). 
We also used this assay to determine which kinin residues are critical for the kinin peptide-receptor interaction. Insect kinin peptides share a C-terminal pentapeptide that represents the minimal sequence required for biological activity, also known as core. In Table 1, the kinin peptide core analogs were synthesized as an Alanine replacement series of the core kinin pentapeptide FFSWGa and tested by a calcium bioluminescence plate assay (Taneja-Bageshwar et al., 2006). We found that the amino acids Phe1 and Trp4 were essential for activity of the insect kinins for both receptors.
The assay also can be used to test peptides designed for enhanced biostability. Table 2 shows the designed kinin analogs containing amino isobutyric acid (Aib) tested on the tick recombinant kinin receptor and Figure 2 shows the activity comparison of six alpha-amino isobutyric acid analogs on the tick kinin receptor expressing CHO-K1 cell line by a calcium bioluminescence plate assay (Taneja-Bageshwar et al., 2009). The hexapeptide analog FFFSWGa is added for a positive control for receptor activity. The analog FF[Aib]WGa resulted more active than this hexapeptide control analog. The analog with two aminoisobutyric acid replacements, [Aib]FF[Aib]WGa, was the most potent of the double-replacement analogs tested (Table 2 and Figure 2).
For more examples of how this assay has been and can be applied see Nachman and Pietantonio (2010), Nachman et al. (2009), Taneja-Bageshwar et al. (2008a), and Taneja-Bageshwar et al. (2008b).
<img src="/files/ftp_upload/2732/2732fig1.jpg" alt="Figure 1"/> 
Figure 1. Estimation of Aedes kinins effective concentration (EC50) on CHO-K1 E10 cells by a calcium-bioluminescence plate assay. The y-axis in the concentration-response curves was obtained from bioluminescence units expressed as a percentage of the maximal response observed for each peptide. Data points represent the average of six replicates obtained during three independent experiments. Bars represent the standard error. (A) Estimation of Aedae-K1 EC50 = 48 nM. (B) Estimation of Aedae-K2 EC50 = 26 nM. (C) Estimation of Aedae-K3 EC50 = 16 nM. EC50 Aedae-K3 &lt; EC50 Aedae-K2 &lt; EC50 Aedae-K1; P &lt; 0.05. Statistical analysis and graphs were with the GraphPad Prism 4.0 software.
<img src="/files/ftp_upload/2732/2732fig2.jpg" alt="Figure 2"/> 
Figure 2. Activity comparison of six alpha-amino isobutyric acid analogs on the tick kinin receptor expressing CHO-K1 cell line by a calcium bioluminescence plate assay. The y-axis represents percent maximal bioluminescence units for each analog expressed as a percentage of bioluminescence observed at a concentration versus the maximal response observed among all concentrations tested for each analog. Statistical analysis and graphs were performed with GraphPad Prism 4.0 software. 
<table border="1">
	<tbody>
 <tr>
 	<td>&nbsp;</td>
 	<td colspan="2">Tick receptor cell line</td>
 <td colspan="2">Mosquito receptor cell line</td>
 
 </tr>
 <tr>	
 	<td>Peptides</td>
 <td>EC50 (nM)</td>
 <td >Maximal bioluminescence response at 1 mM</td>
 <td>EC50 (nM)</td>
 <td>Maximal bioluminescence response at 1 mM</td>
 
 </tr>
 <tr>
 	<td>AFSWGa</td>
 <td>I</td>
 <td>I</td>
 <td>I</td>
 <td>I</td>
 </tr>
 <tr>
 	<td>FASWGa</td>
 <td>586</td>
 <td>5,600 </td>
 <td>N.D. </td>
 <td>400</td>
 </tr>
 <tr>
 	<td>FFAWGa</td>
 <td>64 </td>
 <td>12,800 </td>
 <td>621 </td>
 <td>3,050</td>
 </tr>
 <tr>
 	<td>FFSAGa</td>
 <td>I</td>
 <td>I</td>
 <td>I</td>
 <td>I</td>
 </tr>
 <tr>
 	<td>FFSWAa</td>
 <td>417</td>
 <td>10,600 </td>
 <td>2,800 </td>
 <td>1,830</td>
 </tr>
 <tr>
 	<td>FFSWGa</td>
 <td>590</td>
 <td>10,800 </td>
 <td>N.D. </td>
 <td>525</td>
 </tr>
 <tr>
 	<td>FSWGa</td>
 <td>I</td>
 <td>I</td>
 <td>I</td>
 <td>I</td>
 </tr>
 <tr>
 	<td>FFSWa</td>
 <td>I</td>
 <td>I</td>
 <td>I</td>
 <td>I</td>
 </tr>
 <tr>
 	<td>FFSWG-OH</td>
 <td>I</td>
 <td>I</td>
 <td>I</td>
 <td>I</td>
 </tr>
 <tr>
 	<td>FFFSWGa</td>
 <td>259</td>
 <td>13,000</td>
 <td>562</td>
 <td>10,000</td>
 </tr>
 <tr>
 	<td>FF[Aib]WGa</td>
 <td>29</td>
 <td>12,700 </td>
 <td>445 </td>
 <td>9,300</td>
 </tr>
 
 
 </tbody>
</table>
Table 1. Estimated potencies (EC50) and maximal bioluminescence response of all the peptides tested on tick and mosquito receptor transfected cell lines*.
*The EC50 estimates the concentration required to induce a half-maximal response. I: Inactive if bioluminescence response is less than 300 units (level of vector-only transfected cells). A: The position where the respective residue in the peptide FFSWGa has been replaced by alanine. N.D.: The analog was tested but was either not very active or was not active at lower molarities, thus an EC50 could not be determined.
<table border="1">
	<tbody>
 <tr>
 	<td>K-Aib-1 </td>
 <td>[Aib]FF[Aib]WGa</td>
 
 </tr>
 <tr>
 	<td>K-Aib-2 </td>
 <td>[&#x03B1; MeF]FF[Aib]WGa</td>
 
 </tr>
 <tr>
 	<td>K-Aib-3 </td>
 <td>Ac-R[Aib]FF[Aib]WGa</td>
 
 </tr>
 <tr>
 	<td>K-Aib-4 </td>
 <td>Ac-R[&#x03B2;3F]FF[Aib]WGa</td>
 
 </tr>
 <tr>
 	<td>K-Aib-5 </td>
 <td>[Aib]RFF[Aib]WGa</td>
 
 </tr>
 <tr>
 	<td>K-Aib-6 </td>
 <td>[Aib-Aib-Aib-Aib]RFF[Aib]WGa</td>
 
 </tr>
 

 </tbody>

</table>
Table 2. Kinin analogs (K) containing amino isobutyric acid (Aib) tested on the tick recombinant kinin receptor. Ac: acetyl; &alpha; Me: &alpha; methyl- phenylalanine; &beta;3F: &beta;3 -phenylalanine; a: amide.

<ol>
	<li>Kozak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+5'-noncoding+sequences+from+699+vertebrate+messenger+RNAs.">An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs.</a> Nucleic Acids Research. 15, 8125-8148 (1987).</li><li>Nachman, R., Pietrantonio, P. V., Geary, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+mimetic+analogs+of+insect+kinin+neuropeptides+with+arthropod+receptors.">Interaction of mimetic analogs of insect kinin neuropeptides with arthropod receptors.</a> Neuropeptide systems as Targets for Parasite and Pest Control. , (2010).</li><li>Nachman, R. J., Pietrantonio, P. V., Coast, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+the+development+of+novel+pest+management+agents+based+upon+insect+kinin+neuropeptide+analogs.">Towards the development of novel pest management agents based upon insect kinin neuropeptide analogs.</a> Annals of the New York Academy of Sciences. , 1163-11251 (2009).</li><li>Taneja-Bageshwar, S., Strey, A., Isaac, R. E., Coast, G. M., Zubrzak, P., Pietrantonio, P. V., Nachman, 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=Biostable+agonists+that+match+and/or+exceed+the+activity+of+insect+kinins+on+recombinant+arthropod+GPCRs.">Biostable agonists that match and/or exceed the activity of insect kinins on recombinant arthropod GPCRs.</a> General and Comparative Endocrinology. 162, 122-128 (2009).</li><li>Taneja-Bageshwar, S., Strey, A., Kaczmarek, K., Zabrocki, J., Pietrantonio, P. V., Nachman, 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=Comparison+of+insect+kinin+analogs+with+cis-peptide+bond,+type+VI-turn+motifs+identifies+optimal+stereochemistry+for+interaction+with+a+recombinant+arthropod+kinin+receptor+from+the+southern+cattle+tick+Boophilus+microplus.">Comparison of insect kinin analogs with cis-peptide bond, type VI-turn motifs identifies optimal stereochemistry for interaction with a recombinant arthropod kinin receptor from the southern cattle tick Boophilus microplus.</a> Peptides. 29, 295-301 .</li><li>Taneja-Bageshwar, S., Strey, A., Zubrzak, P., Williams, H., Reyes-Rangel, G., Juaristi, E., Pietrantonio, P. V., Nachman, 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=Identification+of+selective+and+non-selective,+biostable+beta-amino+acid+agonists+of+recombinant+insect+kinin+receptors+from+the+southern+cattle+tick+Boophilus+microplus+and+mosquito+Aedes+aegypti.">Identification of selective and non-selective, biostable beta-amino acid agonists of recombinant insect kinin receptors from the southern cattle tick Boophilus microplus and mosquito Aedes aegypti.</a> Peptides. 29, 302-309 .</li><li>Taneja-Bageshwar, S., Strey, A., Zubrzak, P., Pietrantonio, P. V., Nachman, 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=Comparative+structure-activity+analysis+of+insect+kinin+core+analogs+on+recombinant+kinin+receptors+drom+southern+cattle+tick+Boophilus+microplus+(Acari:+Ixodidae)+and+mosquito+Aedes+aegypti+(Diptera:+Culicidae).">Comparative structure-activity analysis of insect kinin core analogs on recombinant kinin receptors drom southern cattle tick Boophilus microplus (Acari: Ixodidae) and mosquito Aedes aegypti (Diptera: Culicidae).</a> Archives of Insect Biochemistry and Physiology. 62, 128-140 (2006).</li><li>Pietrantonio, P. V., Jagge, C., Taneja-Bageshwar, S., Nachman, R. J., Barhoumi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mosquito+Aedes+aegypti+(L.)+leucokinin+receptor+is+a+multiligand+receptor+for+the+three+Aedes+kinins.">The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins.</a> Insect Molecular Biology. 14, 55-67 (2005).</li><li>Holmes, S. P., Barhoumi, R., Nachman, R. J., Pietrantonio, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+a+G+protein-coupled+receptor+from+the+Southern+cattle+tick+Boophilus+microplus+(Acari:+Ixodidae)+identifies+it+as+the+first+arthropod+myokinin+receptor.">Functional analysis of a G protein-coupled receptor from the Southern cattle tick Boophilus microplus (Acari: Ixodidae) identifies it as the first arthropod myokinin receptor.</a> Insect Molecular Biology. 12, 27-38 (2003).</li><li>Staubli, F., Jorgensen, T. J. D., Cazzamali, G., Williamson, M., Lenz, C., Sondergaard, L., Roepstorff, P., Grimmelikhuijzen, C. J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+of+the+insect+adipokinetic+hormone+receptors.">Molecular identification of the insect adipokinetic hormone receptors.</a> Proceedings of the National Academy of Sciences USA. 99, 3446-3451 (2002).</li><li>Stables, J., Green, A., Marshall, F., Fraser, N., Knight, E., Sautel, M., Milligan, G., Lee, M., Rees, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioluminescent+assay+for+agonist+activity+at+potentially+any+G-protein+coupled+receptor.">A bioluminescent assay for agonist activity at potentially any G-protein coupled receptor.</a> Analytical Biochemistry. 252, 115-126 (1997).</li></ol>

No conflicts of interest declared.

We were able to perform the functional characterization of the first neuropeptide receptor discovered from the Arachnida (ticks, mites and spiders), the tick kinin receptor, using this protocol. This method has three primary applications. First, the technique can be applied for receptor deorphanization through ligand activity measurements. Second, the assay can resolve ligand-receptor structure-activity relationships (SAR). Third, the methods can be used in drug discovery. Furthermore, this protocol can be used to study the activity of agonists or antagonists on almost any GPCR. We are beginning to adapt this protocol for screening of small libraries. The cell line we utilized does not express the ubiquitous G protein G16. We did not need it because arthropod kinin receptors signal through the Gq protein and the intracellular calcium cascade and they conserve this signaling properties in mammalian cells as shown here.

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>DMEM</td>
 <td>Invitrogen</td>
 <td>ABCD1234</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>CHO-K1 cells</td>
 <td>ATCC</td>
 <td>CCL-61</td>
 <td>Manassas, VA, USA</td>
 </tr>
 <tr>
 	<td>F12K medium</td>
 <td>Invitrogen</td>
 <td>21127</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Fetal Bovine Serum</td>
 <td>Sigma-Aldrich</td>
 <td>F0643</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Trypsin-EDTA (10x)</td>
 <td>Invitrogen</td>
 <td>15400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Antibiotic-Antimycotic</td>
 <td>Invitrogen</td>
 <td>15240</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Opti-MEM I 
Reduced Serum Medium
</td>
 <td>Invitrogen</td>
 <td>31985</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Lipofectin Reagent</td>
 <td>Invitrogen</td>
 <td>18292-011</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>GENETICIN</td>
 <td>Invitrogen</td>
 <td>10131035</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>MC1061/P3 Ultracomp</td>
 <td>Invitrogen</td>
 <td>C663-03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>QIAprep spin miniprep kit</td>
 <td>Qiagen Inc.</td>
 <td>19064</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>FuGENE 6 Transfection reagent</td>
 <td>Roche</td>
 <td>11 814 443 001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>96-well white thin bottom microtitere plate</td>
 <td>Costar</td>
 <td>3610</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>calcium-free DMEM media</td>
 <td>Invitrogen</td>
 <td>21068</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Coelenterazine</td>
 <td>Invitrogen</td>
 <td>C-2944</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Bright-Line Hemacytometer </td>
 <td>Hausser Scientific</td>
 <td>&nbsp;</td>
 <td>Horsham, PA</td>
 </tr>
 
 <tr>
 	<td>NOVOstar</td>
 <td>BMG Labtechnologies</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 	<td>Prism software 4.0</td>
 <td>GraphPad Software Inc.</td>
 <td>&nbsp;</td>
 <td>San Diego, CA, USA</td>
 </tr>
 <tr>
 	<td>T-25 and T-75 Flasks</td>
 <td>BD Falcon</td>
 <td>353014 and 353135</td>
 <td>&nbsp;</td>
 </tr>
 
 
 </tbody>
</table>

a calcium bioluminescence assay for functional analysis of mosquito (aedes aegypti) and tick (rhipicephalus microplus) g protein-coupled receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

Watch this Scientific Journal Video about A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors at JoVE.com

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

a-calcium-bioluminescence-assay-for-functional-analysis-mosquito

Research

JoVE Journal

Immunology and Infection

15.6K ビュー.  Texas A&M University (TAMU). このプロトコルは、クローン細胞ラインの選択とその同族のGPCRで合成された節足動物の神経ペプチドの構造活性相関を分析するためにカルシウムの生物発光アッセイの手順について説明します。このアッセイは、受容体のdeorphanizationと合成アナログ設計およびペプチド/薬物リード発見のための構造活性相関の研究にも使用できます。

ビデオ: A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...