Cell colony formation assay

In aneuploid infinite cell lines and cancer cell lines, there are still different cell subpopulations, and their functions and growth characteristics are somewhat different. Some of the subpopulations have greater adaptability to the culture environment and have stronger independent survival. Ability, high cell colony rate. The purified cell population is derived from a common progenitor cell, and its genetic traits and biological characteristics are similar, which is beneficial to experimental research. Primary cultured cells and diploid finite cell lines have low cell colony rates. Cell colony formation rates should be determined prior to cell colony culture to understand the ability of cells to grow at very low density conditions.

At present, only cancer stem cells have the ability to form colonies, and colony inhibition rates are often used in anticancer drug sensitivity tests and tumor radiobiology experiments.

Colony inhibition rate = (1 - (experimental colony formation rate / control colony formation rate)) × 100%

(1) Principle

Cell colony formation rate Single cells proliferate in vitro for more than 6 generations, and the cell population composed of their progeny is called colony or clone. Each clone contains more than 50 cells and is between 0.3 and 1.0 mm in size. The colony formation rate indicates the independent viability of cells. Common methods include a plate colony formation test and a soft agar colony formation test.

(2) Laboratory supplies

1. Material: Hela cells.

2. Equipment: (diameter 60mm) culture dish, cell counting board, beaker, straw, centrifuge, centrifuge tube, waste bottle, inverted microscope, carbon dioxide incubator, ultra-clean workbench, water bath.

3. Reagents: Giemsa dye solution, 0.25% trypsin digest, serum cell culture medium, Aner iodine, agar.

(three) method

1. Plate colony formation assay

This method is applicable to adherently grown cells, including cultured normal cells and tumor cells.

(1) For the exponential growth phase cells, a cell suspension is prepared by a conventional digestion and passage method.

(2) The cell suspension is repeatedly blown to fully disperse the cells, and the percentage of single cells should be above 95%. Count the cells and adjust the cell concentration with the medium for use.

(3) The cell suspension is diluted in proportion according to the cell proliferation ability. Generally, 5 ml of the cell suspension was inoculated into a Petri dish (60 mm in diameter) at a concentration of 50, 100, and 200 cells per dish, and the dish was gently shaken in a cross direction to uniformly disperse the cells.

(4) The culture dish is cultured at 37 ° C, 5% CO 2 for 2 to 3 weeks, and the fresh culture solution is replaced at the appropriate time according to the pH change of the culture solution.

(5) When macroscopic clones appeared in the culture dish, the culture was terminated, the culture solution was discarded, and the PBS solution was carefully washed twice, and air-dried. The methanol was fixed for 15 minutes, and the methanol was discarded and air dried. Stain with Giemsa dye solution for 10 minutes, wash the dye solution slowly with running water, and air dry.

2. Soft agar colony formation test

The method is applicable to cells that are not anchor-dependent growth, such as bone marrow hematopoietic stem cells, tumor cell lines, and transformed cell lines. Using agar solution without adhesion and coagulation, the tumor cells are mixed into the agar solution, and the agar solution is coagulated to place the tumor cells in a certain position. The tumor cells in the agar may move around in all directions, so it can be used to detect tumors. The ability of cells to move actively. Tumor cells can be proliferated in a suitable medium to determine the rate of tumor cell colony formation. Hematopoietic system soft agar colony formation test method is the same, mainly used for research on cell differentiation, but the use of different media.

(1) Same as above (1)~(3).

(2) Adjust the cell suspension density to 1 × 103 cells/ml.

(3) Prepare the bottom agar, completely melted 5% agar and freshly complete culture medium pre-warmed at 37 °C, uniformly mix at a ratio of 1:9 at 40 ° C, and add to a Petri dish (60 mm in diameter) containing 0.5% agar per dish. The medium was 2 ml, and the agar was completely solidified at room temperature.

(4) Prepare the upper agar, transfer 1.5ml of cell suspension with different density gradients at 37°C (containing 50, 100, 200 per dish) into a small beaker, add 40°C, 5% agar and other volumes to mix. 0.25% semi-solid agar medium. The prepared semi-solid agar medium was immediately added to a petri dish covered with a bottom agar, and the agar was solidified at room temperature. The cells were statically cultured at 37 ° C, 5% CO 2 for 2-3 weeks.

(4) Experimental results

1. Regularly observe the formation of colonies during cell culture.

2. Count more than 50 cell clones under the microscope and calculate the colony formation rate as follows:

Colony formation rate (%) = (number of colonies / number of cells inoculated) × 100

(5) Attention

1. Agar is unstable to heat and acid. If it is repeatedly heated, it is easily degraded, produces toxicity, and the hardness of agar decreases. Therefore, the agar is autoclaved (10 lbs and 15 minutes) and then dispensed once.

2. In the cell suspension, the cell dispersion is >95%.

3. When soft agar culture, pay attention to the temperature of the agar mixed with the cells should not exceed 40 ° C, so as not to burn the cells.

4. The density of inoculated cells should not be too high.

5. The cells cultured under low density conditions, the survival rate decreased significantly, and the colony formation rate of the infinite cell lines and tumor cell lines was generally above 10%. However, primary cultured cells and finite cell lines are only 0.5 to 5%, or even zero. In order to increase the colony formation rate, a cell-promoting substance such as insulin or dexamethasone is added to the medium as necessary.

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