Cell culture and growth play an important role in biological laboratories.
The best interest of cell culture is that "cell division" can be carried out in the environment under our control, and can be widely used.
A new cell line must undergo some preliminary analysis and be confirmed as stable before being applied to the test.
Before the test, you must first do plating efficiency test: general theory, a value of 100 cells, 100 community should grow up, but as a result of external factors and the status of the cell itself, in fact less than 100 community;
The percentage of the population that was harvested was the ratio of the number of plant cells to which the cell population was planted, and the natural death of the cells was corrected.
Experimental procedure:
1, will contain 10% or serum replacements DMEM (tested) training of 293 t cells in T75 - flask to 80% confluency.
2. Using trypsin-edta to process the cells, after centrifugation, add a moderate amount of serum or serum replacement DMEM to make cell suspension and measure the cell concentration.
DMEM attenuation cell concentration of no serum or serum was 1 x 102 living cells/ml.
3, will be 1 ml cell suspension into 6 - well cell culture plate, and the other with 1 ml containing different concentrations of serum or serum substitutes (20%, 10%, 4%, 2%, 20%, 0.4%) of the DMEM, make serum final concentration of 10%, 5%, 2%, 1%, 0.5%, 0.2%.
A control trial was performed with a tested serum or serum replacement.
4, at 37 ℃ and 5% CO2 incubator culture 3 ~ 5 days, don't need to change during the culture medium, the colonies to the naked eye, and between communities don't contact with each other.
5, remove medium, add 1 ml Carnoy 's fixed liquid volume ﹦ 3:1) (methanol volume: glacial acetic acid, let stand for 10 min at room temperature.
Remove the fixed fluid and clean twice with secondary water.
6. Add 1 ml 10% Giemsasolution, and stain for 2-3 min at room temperature.
7. Remove the dye, and use secondary water to clean twice.
8. Counting the number of communities under the microscope.
9. Calculation of the SPE (Serum PlatingEfficiency) :
SPE = (No. Of colonies/well) / 100x 100 %
10, calculation RPE (Relative Plating Efficiency) :
Total colonies of six well (test)/total colonies of six well
(control)] x100 %
11. The serum or serum replacement of serum or serum was obtained by comparing the RPE of the serum or serum substitute medium
The effect of surrogate on cell growth.