Measurement experiments of wall affixation efficiency
Measurement experiments of wall affixation efficiency
Inoculate cells at low density, incubate until colonies form, stain and count colonies Source : Animal Cell Culture : A Guide to Basic Techniques 5th Edition
Operation method
Scheme 21.10 Experiments for the determination of walling efficiency
Principle
Cells were inoculated at low density, incubated until colonies formed, stained and counted for colonies Move makings For more product details, please visit Aladdin Scientific website.
non-sterile
Growth solution 400 ml
0.25% crude trypsin 10 ml
Petri dishes, 6 cm 20 pcs.
Petri dishes, 6 cm 20 test tubes or general purpose glass containers for dilution
Non-sterile
Blood counting plate or electronic counter
Fixative: anhydrous methanol 100 ml
D-PBSA 200 ml
Dye: crystal violet 100 ml
Filter hopper and filter paper (to filter the dye)
Procedure:
1. trypsin digest the cells (see Scheme 13. 2) to make a single cell suspension.
2. While the cells are being digested.
(a) Number on the bottom of the Petri dish;
(b) Calculate the amount of culture solution required for each dilution step (Fig. 21.10). (b) Calculate the amount of culture solution needed for each dilution step (Fig. 21.10).
3. When the cells become rounded and begin to float.
(a) Disperse the monolayer in culture medium containing serum or trypsin inhibitor;
(b) Cell counting;
(c) Dilution of cells to:
(i) 2X104 cells/ml into two 25 cm2 culture flasks for routine maintenance.
ii) The maximum concentration of the diluent is 2X103 cells/m l.
iii) Dilute ii) to 5 concentrations, 200, 100, 50,20 and 10 cells/ml.
4. Five concentrations(iii) of cells were inoculated in 5 ml of culture medium in Petri dishes. An additional 2 X 103 cells/ml were added to two 6cm Petri dishes as a control in case the clonal culture was unsuccessful (at least to ensure that there were cells in the maximum dilution of the culture solution).
5. Fill the Petri dishes with 5 % CO2 and place in a warming oven.
6. Place the Petri dish in a clear plastic box, preferably in a humid CO2 incubator with a limited channel population and used only for cloning experiments.
7. incubate for 1 to 3 weeks until colonies are visible to the naked eye.
8. Stain cells with crystal violet:
(a) Aspirate the culture solution from the petri dish;
(b) Wash the cells with D-PBSA and discard the washings;
(c) Add 5 ml of fresh D- PBSA, then add 5 ml of methanol and mix gently (be careful to avoid colonies floating up);
(d) Replace the methanol D-PBSA mixture with 5 ml of fresh methanol (50:50) and fix the cells for lO min;
(e) Discard the methanol and add filtered crystal violet, 2-3 ml per 6 cm dish, making sure that the entire growth surface is covered by the dye;
(f) Stain for lO min.
(h) Remove the dye, filter and pour into the mother liquor bottle.
9. Count the number of colonies in each IE, do not count colonies with less than 50 cells. It is easier to count under magnification. It is important to set a threshold above which a colony can be counted, and if the majority of colonies are between 100 and several f cells, the threshold can be set at 50 cells per colony. In operation, counting with the naked eye is common. However, when the number of colonies is very small (<100 cells), the threshold can be set at 16 cells per colony. below 16 cells, which corresponds to 4 consecutive cell divisions, it is difficult for more cells to proliferate.