Experiments on colony formation of cells

Summary

Hematopoietic growth factors is a general term for a class of cytokines that mainly act in the hematopoietic system. The survival, proliferation and differentiation of hematopoietic cells cannot be achieved without hematopoietic growth factors in the hematopoietic microenvironment. Therefore, the detection of hematopoietic growth factors is essential in the study of hematopoietic process. Compared with ELISA and cell proliferation assay, colony formation assay can not only reflect the level of cytokines, but also reflect the effect of different growth factors on colony formation.

Operation method

Experiments on colony formation of cells

Materials and Instruments

Equipment:
① 35 mm cell culture dish (for suspension of cells)
② 10 cm cell culture dish
③ Surgical instruments
④ 1 mL syringe
Reagents:
① Methyl cellulose semi-solid medium MethoCult (Stem Cell Technology 03334).
① Methyl cellulose semi-solid medium MethoCult (Stem Cell Technology 03334); ② 1x PBS (NaCl 137 mmol/L, KCI 2.7 mmol/L, Na2); ③ 1 mL of syringe.
NaCl 137 mmol/L, KCI 2.7 mmol/L, Na2
HPO, 10 mmol/L.

Move

The basic procedure of colony formation experiments with cells can be divided into the following steps:

A Fetal hepatocytes are harvested under sterile conditions and separated into single cell suspensions using a syringe. B Cell counting. B Cell counting. C Preparation of the cell suspension at a concentration of 10x the final cell inoculum density. D For experiments with 2 sub-wells, mix 0.3 mL of cell suspension with 3 mL of methylcellulose medium. E Dispense 1.1 mL of methylcellulose cell culture solution per 35 mm dish. F Place two 35 mm dishes and one uncovered 35 mm dish in each 10 cm dish. Add 3 mL of sterilized water to the uncovered 35 mm dish to moisturize and reduce the risk of contamination. G Place the petri dish at 37 °C, 5% _CO2, and humidity >95%. H Hematopoietic clones can be counted under a microscope after 2 days for CFU-E and 7 to 10 days for BFU-E.

I Figure 8-8-2 shows the number of BFU-E, CFU-GM, and CFU-GEMM in each tissue under normal conditions.

Caveat

1. Melt methylcellulose medium overnight.

2. operate aseptically and sterilize reagents and consumables beforehand.

3. Cells can also be screened by magnetic beads or FACS sorting, etc., and then planted on Methylcellulose Medium.

4. In order to maintain the activity of stem ＼ progenitor cells, the whole procedure should be as fast as possible to minimize the damage to cell activity.

5. Different cytokines can be added to the methylcellulose to examine the ability of the stem/progenitor cells to differentiate into different terminally differentiated cells; StemCell Technologies has methylcellulose medium with different growth factors for different experimental purposes.

6. Benzidine staining can be used to help identify BFU-E and CFU-E clones.

7. Figure 8-8-1 shows the appearance of other clones for identification.

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