Steps for detecting cell survival rate

Cell activity testing aims to understand the survival status and activity of cells, and is it suitable for subsequent experiments. Poor cell activity is also one of the factors that lead to unsuccessful transfection in virus transfection experiments. It is also used for drug research, which extends the study of the smallest living organism to the understanding of the functional drug characteristics of the entire body. So, how to do cell activity testing? The following is one of the manuscripts we have compiled technically, focusing on Qida Biology and not getting lost in the experimental operation:

This operation step adopts PicoGreen 96 well plate technology. This method is used to evaluate the sensitivity of different tumor cell lines to chemotherapy drugs.

reagent

Medium containing 10% FBS

PicoGreen

TryLE express (Trypsin) (Qida Biological, product number: SD0003)

Phosphate buffered saline (PBS) (Qida Biological, product number: SD0029)

deionized water

Consumables

96 well culture plate

TECAN Genios Instrument (PHENIX)

Thermostat

Metal sheet

Spectrophotometer

1. Extract the culture medium and wash once with 5 ml PBS. Add 2ml tryLE express. After cell separation, add 10ml of complete culture medium. Use 5-10ml pipette to transfer the cell suspension to make single cell suspension. This step is very important (to obtain the single cell suspension, place the tip of the pipette at the bottom of the flask, and then push the cell suspension out of the pipette).

2. Count cells and dilute to 1 x 10 ^ 5/ml, place cells at 100 μ In a 96 well plate with 1 x10 ^ 3/well.

3. Dilute the drug of interest into a series of concentrations and add 100% μ Add an amount of l into the well (the drug is prepared at twice the final concentration).

4. Incubate the orifice plate in an environment of 37 ° C and 5% carbon dioxide for one week, during which the liquid needs to be changed according to the normal frequency,Gently suck off the plate culture medium. Then wipe to remove the remaining culture medium.

5. Using PBS 200 μ Wash twice per well. Gently wipe off PBS by shaking the orifice plate.

6. Add 100 to each hole μ Deionized water. Place the culture plate in a 37 ° C, 5% CO2 incubator for 1 hour.

7. Dilute PicoGreen with 1:200 in deionized water (requires 100 μ L/well). Be sure to calculate how much you need. Wrap the plate in foil and let it sit at room temperature for 1 hour (if you are busy, this step can be completed overnight).

8. Measure the fluorescence intensity of the board using a spectrophotometer.

Processing data:

Survival rate=average fluorescence intensity of the experimental well x 100%

Average fluorescence intensity in the control well

Note: PicoGreen is a fluorescent dye that selectively binds to dsDNA, and its properties are similar to SYBR Green I. It has maximum excitation at 480 nm and emission at 520 nm.

References/参考文献：

1. Ahn, S. J., Costa, J. and Emanuel, J. R. (1996). PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR. Nucleic Acids Res 24(13): 2623-2625.

2. Enger, O. (1996). Use of the fluorescent dye PicoGreen for quantification of PCR products after agarose gel electrophoresis. Biotechniques 21(3): 372-374.