M. Fero 11/20/2011
BSA in IMDM (100 mg/mL = 10% w/v)
2.6% Methylcellulose in IMDM (StemCell Technologies #H4100)
Growth Factor Mix
Heat inactivated FBS
|Growth Factor Mix||CFC-Mix||GM-CFC||G-CFC||BFU-E||CFU-E|
|IMDM||38% v/v||40% v/v||40% v/v||8% v/v||49.6% v/v
(or 9.6% for MC)
|FBS||20% v/v||-||-||20% v/v||20% v/v|
|Horse serum||-||20% v/v||20% v/v||-||-|
| BSA in IMDM
|10% v/v||±10% v/v||±10% v/v||10% vol||10% vol|
|WEHI-3B C.M. or IL-3 (100 ng/mL)||10% v/v||-||-||10% vol||-|
|hEpo 50 U/mL||2% v/v||-||-||2% vol||0.4% vol|
|10% v/v||10% v/v||10% v/v||10% v/v||10% v/v|
|3.3% agar||10% v/v||10% v/v||10% v/v||-||10% v/v|
|-||-||-||50% v/v||(or 50% v/v)|
(Kaushansky and Broudy add the following: ß-ME (50 µM final conc.), 1% Pen/Strep/Fungizone, see Cell 1996 ref.)
10x Cell Suspensions:
|Source||Bone Marrow||Spleen||Bone Marrow after
1. Prepare cell suspensions in IMDM + 2% FBS. Use heat inactivated FBS if antibody staining will be done. It is not necessary to remove RBC from spleen or marrow. Cells may be kept at 4°C for several hours.
2. Meanwhile melt agar in a boiling water bath and then place in 55°C bath until ready for use.
3. Add 0.5mL (1/10 of final volume) of cell suspension to 4 mL Growth Factor Mix in a disposable tube and warm to 37°C.
4. To add the agar pipet it up/down in prewarm the tip. Add 0.5 mL of the melted agar to the cell/GF mix with continous vortexting and quickly pipet 1.5 mL into three wells of a 6-well plate or 35mm dishes.
5. Cool the dish at 4°C for 2-3 minutes to make the agar set.
1. Prepare cell suspensions in IMDM + 2% h.i. FBS. It is not necessary to remove RBC from spleen or marrow. Cells may be kept at 4°C for several hours.
2. Add 0.5 mL (1/10 of final volume) of cell suspension to 2 mL Media/Growth Factor cocktail in a disposable tube and warm to 37°C.
3. Add an equal volume (2.5 mL) of methyl cellulose/IMDM to the cell/GF mixture and plate ~1.5 mL into three wells of a 6-well plate or into 35mm dishes.
Incubate dishes in a humidified incubator at 36ºC with 5% CO2 and hypobaric oxygen (5% O2). The CO2 maintains the pH of the media and the lower temperature and low oxygen promote growth of hematopoietic cells. The low oxygen also helps promote hemoglobinzation of erythroid cells. To minimize nitrogen utilization the incubator should only be gassed after the plates have been placed inside. Also the incubator should only be opened briefly to retrieve plates on the day(s) that they are counted.
|Day||7 - 12||7||7||5 - 6||2|
|Appearance||>50 cells||>50 cells||>50 cells||200 - 10,000 cells in 3-8 clusters||6 - 60|