The cells were treated with different concentrations of experimental reagents for 24h. After trypsin digestion, the cells are inoculated at low cell density, incubated for 1-3 weeks, and stained to count the number of colonies. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition.
Operation method
Scheme 22.3 Clone formation experiments with walled cells
Principle
The cells were treated with different concentrations of experimental reagents for 24h . After trypsin digestion, the cells were inoculated at a low cell density, incubated for 1 to 3 weeks, and stained (see Color Fig. 6a,e) to count the number of colonies (Fig. 22.1). Move Materials For more product details, please visit Aladdin Scientific website.
Aseptic
Growth solution
D-PBSA
0.25% crude trypsin
Compounds to be tested are prepared at 10 times the maximum concentration to be used and dissolved in serum-free growth solution; pH and osmolality of the test solution are checked and adjusted if necessary;
Culture flask, 25cm2
Petri dish, 6cm or 9cm, mark the sides of the bottom dish
non-sterile
D-PBSA
Methanol
1 % Crystalline Violet
Blood counting plate or electronic cell counter
Procedure
1. Prepare a series of cells cultured in 25 cm2 flasks, 3 flasks of each of the 6 reagent concentrations and 3 controls. Inoculate each flask with 4.5 ml of cell suspension (cell density 5x104 cells per ml of growth solution) and incubate for 48h, when the cells will enter the logarithmic growth phase (see section 21.9.2).
2. Prepare a series of dilutions of the substance to be tested, 2 ml of each, to 10 times the desired final concentration:
(a) For compounds being tested for the first time, use a 5-fold dilution in the 3 to 5 logarithmic growth range;
(b ) If the approximate toxic concentration can be predicted, then choose a narrower arithmetic range, with each group separated by 10 or 20 orders of magnitude.
3. Add 0.5 ml of a 10-fold dilution of the compound to be tested to each of the three culture flasks for each concentration group. Start with the control group (without the substance to be tested) and proceed from the lower concentration to the higher concentration.
4. Return the culture bottles to the incubator.
5. If the action of the compound is slow or partially reversible, repeat step 3 twice, i.e., expose the cells to the reagent for 3 days, changing the medium and the compound every day so that the medium and the compound to be tested can be renewed. For fast-acting compounds, a 1-h exposure is sufficient.
6. Pour off the growth solution from each group of 3 bottles in turn (from lowest to highest concentration) and digest the cells with trypsin.
7. Dilute the cells to the density required for clonal growth (see Scheme 14.1) and inoculate them in Petri dishes. Dilute all cells in the same amount as the control, from the highest concentration culture flask to the lowest concentration group, and finally the control.
8. warm the cells until colonies form.
9. Fix cells with anhydrous ethanol and stain with 1% crystalline violet for 10 min (see Scheme 16.3).
10. Wash the dish with tap water, remove the water and invert to dry.
11. Count colonies of more than 50 cells (more than 5 generations). Analysis of growth curves
12. Calculate the cell attachment efficiency at each set of drug concentrations.
13. Calculate the relative apposition efficiency: apposition rate at each set of concentrations/apposition rate in the control group, which is the cell survival fraction.
14. Plot the cell survival fraction at logarithmic or linear drug concentrations, depending on the concentration range used (Figure 22.2).
Determine the IC50 or IC90, which are the compound concentrations at which 50% or 90% of colony formation is inhibited, respectively. Since this is a semi-logarithmic plot, the IC90 is more applicable because it is more likely to fall in the linear portion of the curve, whereas the IC50. tends to fall in the curved portion of the curve so that it is less stable.
16. analysis of sensitivity difference curves:
(a) Slope of the curve and length of the curved portion. A shallower slope and/or a longer bending portion of the curve indicates a decrease in sensitivity; a steeper slope and/or a shorter bending portion indicates an increase in sensitivity. The length and slope of the curved portion have an effect on both IC50 and IC90, with a more significant difference observed at IC90 (Figure 22.3), and IC90 is often taken as a simple derivative;
(b) Resistance fraction. The cellular resistance fraction is the flat portion at the lower end of the curve;
(c) Lack of a curve gradient indicates that all cells are resistant;
(d) Area under the curve: complex survival curves can be compared by calculating the area under the curve, but this is done only for convenience and is not very well founded mathematically.
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