The overall goal of this simple in vitro culture method is to elucidate underlying mechanisms of tumor malignancy under an acidic tumor microenvironment. This method can help answer key questions in the tumor biology field, such as the underlying mechanism of cancer progression and malignancy in response to the tumor microenvironment. The main advantages of this technique are the stability of the medium pH and the ability to compare several acidic pH media.
The implication of this technique extended toward therapy of not only cancer but also as acidic disorders because acidosis can be associated with various types of disease. To begin, dissolve 4.75 grams of DMEM power with L-glutamine in 500 milliliters of water. Also prepare a 0.33-molar sodium bicarbonate solution in a pressure-tight bottle.
Autoclave the medium and solution. After allowing these buffers to cool to 25 degrees Celsius, add five milliliters of 200 millimolar L-glutamine, five milliliters of penicillin-streptomycin, and 50 milliliters of fetal bovine serum to 500 milliliters of DMEM without bicarbonate. For a control medium, add 2.4 milliliters of 0.33-molar sodium bicarbonate solution per 100 milliliters of DMEM without bicarbonate.
For the low-pH medium, add 0.6 milliliters of 0.33-molar sodium bicarbonate solution per 100 milliliters of DMEM without bicarbonate. Next, add 74 microliters of lactate solution to 100 milliliters of control medium to prepare the lactate-induced acidic medium. To prepare the HCl-induced acidic culture medium, add 125 microliters of one-molar HCl to 100 milliliters of control medium.
Check the medium pH after 24 hours of incubation at 37 degrees Celsius under 5%carbon dioxide. Collect the culture medium immediately after removing the culture dish from the incubator. Keep the medium temperature at 37 degrees Celsius using a water bath if necessary.
Measure the pH using a pH meter three times independently. Start cultivating the cells at 37 degrees Celsius under 5%carbon dioxide at least one week before performing experiments. Wash the cells by adding five milliliters of PBS.
Aspirate the PBS, and add a detaching enzyme, such as trypsin. Then, incubate the cells at 37 degrees Celsius until the cells are rounded and start to detach. Next, add five milliliters of culture medium to the detached cells, and deactivate the enzyme.
Transfer the cell suspension to a 15-milliliter tube, and centrifuge for three minutes at 300 times g. Aspirate the supernatant, and resuspend the cells with culture medium. Count the viable cells using a hemocytometer and Trypan Blue.
Seed 0.5 million cells per well in a 10-centimeter dish in 10 milliliters of medium. Then, incubate the cells for 24 hours at 37 degrees Celsius under 5%carbon dioxide. Following incubation, aspirate the culture medium, and wash the cells with five milliliters of PBS.
Then, add 10 milliliters of the acidic culture medium. Incubate the cells for 24 hours at 37 degrees Celsius under 5%carbon dioxide. Aspirate the culture medium, and wash the cells with five milliliters of PBS.
Disrupt the cells by adding RNA extraction reagent and scraping the dish briefly. Then, remove the RNA extraction reagent with a pipette, and deposit the RNA extraction reagent cell lysate into a 1.5-milliliter tube. Leave the tube at room temperature for five minutes.
Add 250 microliters of chloroform, and shake the tube vigorously for about 15 seconds. After leaving the mixture at room temperature for five minutes, centrifuge at least 8, 000 times g for five minutes. Carefully remove the aqueous phase using a pipette.
Place the removed aqueous phase into another 1.5-milliliter tube. Leave behind some of the aqueous phase. Next, add 550 microliters of isopropanol to the aqueous phase, and mix gently before leaving it at room temperature for five minutes.
Then, centrifuge the sample at maximal speed for 20 minutes at four degrees Celsius. Next, pour off the isopropanol, and add one milliliter of 75%ethanol in DEPC-treated water. Mix gently, and centrifuge at 8, 000 times g for five minutes.
Pipette off the ethanol, and let the pellets air-dry. Add approximately 15 to 25 microliters of DEPC-treated water to the RNA pellet. Mix thoroughly.
Finally, measure the concentration of isolated RNA by measuring the optical density at 260 nanometers using a spectrophotometer. In the PCR reaction tube, combine the components listed in the text protocol. Mix and briefly centrifuge the components using a small benchtop centrifuge.
Then, denature the sample RNA and primer for five minutes at 65 degrees Celsius. Spin briefly using a small benchtop centrifuge, and put the sample promptly on ice for at least one minute. Then, add four microliters of 5X reverse transcription buffer, one microliter of reverse transcriptase, and one microliter of RNase inhibitor.
Cap the tube, mix, and then briefly centrifuge the contents using a small benchtop centrifuge. Incubate the combined reaction mixture at 50 to 55 degrees Celsius for 10 minutes. Then, inactivate the reaction by incubating at 80 degrees Celsius for 10 minutes.
To measure the acid response of gene expression using real-time PCR, combine the components listed in the text protocol in a 384-well reaction plate. Then, set up the experiment in the PCR program on a real-time PCR system, as detailed in the text protocol. Finally, place the plate in the qPCR instrument, and start the program.
Expressions of acidic pH responsive IDI1, MSMO1, and INSIG1 mRNAs in PANC-1 cells and AsPC-1 cells was determined by quantitative real-time polymerase chain reaction analysis under control, low-pH lactic acidosis, and HCl-mediated acidosis conditions for 24 hours. Expression of these genes was highly increased under low pH in medium where the acidic pH was caused by reduced bicarbonate levels, as well as in medium where the acidic pH was caused by the addition of lactate or by the addition of HCl. While attempting this procedure, it is important to remember to autoclave bicarbonate frequently, as bicarbonate easily becomes flat.
Also, remember to check the actual medium pH and color. The color of the control medium is darker than that of the acidic pH mediums. After its development, this technique paved the way for researchers in the field of tumor biology and acidosis-related diseases to explore responsible mechanism to acidic microenvironments.
