Currently producing a thesis for establishing Sf21 and High Five cell lines and have to produce assays testing effects of temp, pH, glucose concentration and lactate production within 96 well plates. What assays are people currently employing to test these effects?
@justinhayes
For temperature testing, I recommend a cell proliferation curve accompanied by a cell viability test for 1 week under different temperatures: 23 ° C, 28 ° C and 35 ° C and 37 ° C. The temperature with the best proliferation over time and the best viability is the most viable temperature. The temperature suggestions are hypothetical, I suggest looking in the literature for similar crops to better measure the temperature.
To analyze glucose consumption I suggest 2 methods: 1 - use a colorimetric glucose test kit and check glucose consumption for a period of 3 hours (for example). Distributors: Merck, Laborclin, Labtest. A second test would be the use of a fluorescent glucose analog - 2-NBDG - that you can use to ascertain the uptake of glucose, this method you find in (ZOU; WANG; SHEN, 2005).
for pH measurement you can use pH indicator tape (however it is less accurate). use of a portable pH meter inside the laminar flow cabinet with a fine electrode to submerge in the plate well. For pH, I recommend making a culture especially in a T25 bottle, as the volume is larger and easier to measure in pHmeter. Just repeat the same conditions: Time, temperature, concentration of O2 and CO2, and supply of nutrients equal to those performed on the microplate.
For lactate, you can use the lactate colorimetric assay kit or do the enzymatic activity of Lactate dehydrogenase using commercial kit as well. distributors: Sigma-aldrich, Merck.
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