A cluster of cells formed by proliferation of an ancestral cell in an in vitro culture medium is called a colony. Tumor cells can multiply indefinitely, so they have this ability, while mature and differentiated cells cannot form colonies.
Experimental Methods Basic Protocol Experimental Method Principle HL-60 cells are acute promyelocytic leukemia cells that can form colonies in soft agar medium without the addition of stimulating factors in vitro. Dimethyl sulfoxide is a cell differentiation inducer. HL-60 cells treated with dimethyl sulfoxide are differentiated and differentiated by granulocyte pathway, and the proliferation of cells is reduced. Almost all cells lose their formation in soft agar. The ability to colonize. Therefore, this method can be used for basic research of cell differentiation and efficacy test of clinical tumor treatment.
Experimental material HL-60 cells
Reagents , kits, calf serum, RPMI1640, culture medium, trypan blue agar dimethyl sulfoxide
Instrument , consumables, clean bench, carbon dioxide incubator, microscope, culture bottle, pipette, alcohol lamp, blood cell counting plate, multi-well plate
Experimental Procedure 1. Collect HL-60 cells in logarithmic growth phase, first measure cell viability according to experiment thirteen, and then adjust cell concentration to make cell suspension of 300~1 000 viable cells/ml.
2. Add 9 ml of each flask to each well and adjust the concentration of the cell suspension. Then add 1 ml of 3% agar (melted, placed in a 65 ° C water bath), quickly add and mix.
3. Pipette 140 μl of dimethyl sulfoxide (MDSO) into a vial with a micropipette and mix thoroughly to form the experimental group. The other flask is DMSO-free and is a blank control group.
4. Take a 16 mm multi-well plate, add 1 ml per well (2 ml of 35 mm culture dish) cell suspension, do not have air bubbles, add the experimental group and the control group into two groups, cover and Make a mark.
5. Allow the cell agar suspension to solidify by allowing to stand at room temperature for 20 minutes.
6. Then incubate the plate or plate in a CO2 incubator at 37 °C.
7. Incubate for 7-10 days, visually observe and count cell colonies.
8. Results: Cell clusters (containing more than 500 cells) visible to the naked eye were used as a standard for counting colonies. The HL-60 cells in the control group grew well in the soft agar medium containing 0.3%, and multiple colonies were observed in each well. In the experimental group, HL-60 cells were induced to differentiate by dimethyl sulfoxide, and the cells were soft. Colony formation in agar was significantly reduced.
9. Counting and calculation of colonies:
(1) Number of colonies = sum of cell colonies in n wells / n pores
(2) Colony formation rate = number of colonies / total number of cells inoculated × 100%
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