Soft agar colony culture experiments of tumor cells can be used for (1) basic research on cell differentiation; (2) efficacy testing of clinical tumor therapy, etc.
Operation method
Soft agar colony culture assay of tumor cells
Principle
A cluster of cells formed by the proliferation of one progenitor cell in an in vitro medium is called a colony. Tumor cells have this ability because they can multiply indefinitely, whereas mature differentiated cells cannot form colonies.HL-60 cells, an acute promyelocytic leukemia cell, can form colonies in soft agar medium in vitro without the addition of stimulatory factors. Dimethyl sulfoxide is a cell differentiation inducer, and HL-60 cells treated with dimethyl sulfoxide were directed to mature differentiation according to the granulopoietic pathway, while the proliferative power of the cells was reduced, and almost all of the cells lost the ability to form colonies in soft agar.
Materials and Instruments
HL-60 Cells Move 1. Collect HL-60 cells in logarithmic growth phase, firstly, determine the cell viability according to the experiment XIII, then adjust the cell concentration, and make the cell suspension of 300~1,000 live cells/ml. Caveat 1. Sterilization of glass pipettes and glass culture flasks: 1) autoclave sterilization for more than 15 minutes, 2) dry baking sterilization at 140 degrees for more than 2 hours. 2. Digestive solution (pH7.8) or other additives should be autoclaved or filtered by disposable sterile filter to remove bacteria, and stored at -20 degrees Celsius in separate packages. 3. The aseptic bench should be cleaned first and then wiped with 75% alcohol, and the ultraviolet radiation should be irradiated for more than 40 minutes; all kinds of culture plates should be irradiated for more than 3 hours. 4. culture medium (pH7.2) and serum prepared to do aseptic test: the serum will be added to the culture medium according to 10%, with a sterile glass centrifuge tube or glass bottle to take 5-10ml of complete culture medium in the incubator incubator for 2-3 days, the naked eye to see no turbidity or precipitation and other foreign matter. After the separation and storage at 4 degrees. 5. The incubator should be cleaned with water and then wiped with 75% alcohol (such as ultraviolet light should be irradiated for more than 1 hour, such as autoclave sterilization should be in accordance with the procedures to bacteria), at least once a month. 6. into the operation should pay attention to the first wash hands and wrists, and then wiped with 75% alcohol, operation, pay attention to the level of space in the aseptic table. Hands and objects should not come and go above the mouth of exposed bottles. If the quantity is large, tumor cell culture bottles should be placed parallel to the alcohol lamp to facilitate the operation, and bottles should be separated from each other by a certain distance, and bottles (lids) should be opened by wiping them repeatedly with 75% alcohol or burning them with lamps, and the lamps should be used to burn the mouths first after opening, and then burn the lids. For more product details, please visit Aladdin Scientific website.
Calf serum RPMI1640 culture medium Teparan agar Dimethyl sulfoxide
Ultra-clean bench, Carbon dioxide incubator, Microscope, Culture flasks, Straws, Alcohol lamps, Hematocrit plates, Multi-well plates.
2. Take two culture flasks and add 9 ml of adjusted concentration of cell suspension in each flask, then add 1 ml of 3% agar (melted and placed in 65℃ water bath) to each, add it quickly and mix well.
3. Pipette 140 ul of Dimethyl Sulfoxide (MDSO) with a micro-sampler, add it to one bottle, mix well, for the experimental group, and the other bottle without DMSO, for the blank control group.
4. Take a 16 mm multi-well culture plate, add 1 ml (2 ml for 35 mm dish) of cell agar suspension to each well, do not have air bubbles, add the experimental group and the control group into two groups, cover the lid and mark.
5. Leave at room temperature for 20 minutes to solidify the cell agar suspension.6. Transfer the plates or petri dishes to a CO2 incubator at 37℃ for incubation.
7. Cultivate for 7~10 days, observe and count the cell colonies by naked eye.
8. RESULTS: Cell clusters visible to the naked eye (containing more than 500 cells) were used as the standard for counting colonies. The HL-60 cells of the control group grew well in soft agar medium containing 0.3%, and multiple colony formations were visible in each well, whereas the HL-60 cells of the experimental group showed significantly fewer colony formations in soft agar due to dimethyl sulfoxide-induced differentiation of the cells.
9. Counting and calculation of colonies:
(1) Number of colonies = sum of number of cell colonies in n wells/n wells
(2) Colony formation rate = number of colonies/total number of inoculated cultured cells x 100%.