Bone marrow cells were suspended with agar or methylcellulose, and then the cells were inoculated in Petri dishes with appropriate growth factors.
Operation method
Hematopoietic cell colony formation assay
Principle
Bone marrow cells were suspended with agar or methylcellulose, and then the cells were inoculated in Petri dishes with appropriate growth factors.
Materials and Instruments
Bone Marrow Cells Methyl Cellulose Noble Agar FBS Growth Factor 10% BSA Wehi-CM Move Preparation of Methylcellulose Solution: For more product details, please visit Aladdin Scientific website.
Alpha Medium Petri Petri dishes Water Bath Incubator Incubator
(a) Weigh 7.2 g of methylcellulose into a 500 ml bottle containing a large magnetic bar;
(b) Autoclave the methylcellulose;
(c) Wet the methylcellulose by adding 400 ml of sterile ultrapure water heated to 90°C;
(d) Stir overnight at 4°C to dissolve the methylcellulose and obtain 2x methylcellulose. Dispensing methylcellulose with a syringe without a needle is more accurate than with a pipette.
Alpha culture stock solution preparation:
(a) Powdered Alpha Medium (GIBCO, 10 L packaging volume);
(b) 100 ml 100xMEM Vitamin Stock Solution;
(c) 200 mg Gentamycin Sulfate.
Stir the culture solution on a heated stirrer until the medium is dissolved. The temperature should not exceed 37°C . Then, add ultrapure water to make 3 L . Before final filtration through a filter with a pore size of 0.22 um, it is best to filter successively through filters with pore sizes of 5 um , 1.2 um , 0.8 um and 0.45 um . Dispense into 21 ml and store at -20°C.
Preparation of 2xAlpha Culture Solution:
(a) 21 ml of Alpha culture storage solution;
(b) 25 ml of Fetal bovine serum (FBS);
(c) 1 ml of glutamine (200 mmol/L);
(d) 3 ml of 7.5% sodium bicarbonate.
The above components were placed in a sterile vial, mixed, and equilibrated to 37°C.
I. Cultivation of BFU-E, CFC-mix and CFU-GEMM
1. Mix 2xAlpha culture solution with added BSA and equal amount of 2xmethylcellulose to make the culture solution needed for the experiment. Keep the mixture in a cold state. Methylcellulose is thinner when in the cold state.
2. Prepare cells in triplicate, but prepare as much of the culture mix as is needed for 4 petri dishes. Some of the methylcellulose is often lost due to it sticking to the walls of the test tubes.
3. Add the desired concentration of growth factor to the test tubes. 
4. Add 5x104 cells.
5. Add 1x culture solution to bring the total up to 4.4 ml. Place the test tubes on a vortex mixer and mix.
6. Using a syringe, inject 1 ml of cell suspension into a 3 cm non-culture grade dish.
7. Prepare the cells in triplicate at 37°C under humidified gas conditions with 10% CO2 and 5% O2 under humidified gas conditions for 8~15 d.
Clone Identification
BFU-E colonies can be either a single colony consisting of very small cells or multicentric colonies (like a bomb blast), each with tightly packed very small, pale red or red cells. These colonies occur at a rate of 40 to 80 cells/105 myeloid cells.
CFC-mix colonies are single, dense colonies, usually with a halo of cells that vary widely in size. The colonies can be multicentric, but the cells are clearly multidimensional.CFC-mix colonies occur at a rate of 100-180 cells/105 myeloid cells, with about 10 % of the colonies being mixed colonies containing erythroid lineage cells. If the erythroid lineage cells are not red, it is difficult for the inexperienced to accurately identify these colonies. The colonies should be photographed and the cells removed, then centrifuged to fix the cells and stained with 10% Giemsa. This will help to identify the colony by identifying the cells.
II. Culture of GM-CFC
1. Use 30 mm Petri dishes (non-culture grade). Prepare Petri dishes and label.
2. Prepare 0.3 % agar culture medium and place at 37°C.
3. Prepare bone marrow cells. Count nucleated cells by staining with methylene blue or lysing the cells with Zapo-globin (Beckman Coulter) and then counting the nuclei with an electronic cell counter.
1. 0x107~1. 5x107 cells were obtained from each femur.
4. 0.1 ng/ml rMurGM-CSF was added to each culture dish.
5. 1 ml of agar culture medium containing 7. 5x104 cells was added. Then, shake gently enough to mix the cells and agar. Allow the agar to sink at room temperature.
6. Alternatively, use 0.8 % Methylcellulose Culture Solution. When cultured in methylcellulose medium, it generally produces tighter colonies. This kind of colony is easy to count.
7. Put the petri dish into a clean plastic box.
8. Put the plastic box into a humid incubator, and incubate it at 37℃ and 5 % O2 for 6 d.
9. Count the colony containing 50 cells with an inverted microscope, and express it as the number of colonies in 105 implanted cells.
10. The incidence of GM-CFC in normal bone marrow is 100~120 cells/105 cells. bone marrow cells.