Hematopoietic Colony Forming Cell Assays
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M. Fero 8/20/2004
BSA in IMDM (100 mg/mL = 10% w/v)
To 5 gm BSA (Sigma, A1933)
q.s. to 50 mL with IMDM
Rotate at 4ºC for 1 hr. to dissolve.
q.s. to 50 mL with IMDM
Rotate at 4ºC for 1 hr. to dissolve.
2.6% Methylcellulose in IMDM (StemCell Technologies #H4100)
Add glutamine prior to use
Alternatively it can
be made as per Appendix 6 (p. 277) in Haemopoiesis:
A practical Approach, NG Testa and G Molineux.
Growth Factors
human Erythropoeitin (e.g. Epoetin
Alfa, 2000 U/ 1 mL). Dilute to 50 U/mL
murine GM-CSF (Peprotech)
human G-CSF (e.g. filgrastim, 300 µg / 1 mL). Dilute to 100 ng/mL
murine IL-3 (Peprotech) or WEHI conditioned medium.
murine GM-CSF (Peprotech)
human G-CSF (e.g. filgrastim, 300 µg / 1 mL). Dilute to 100 ng/mL
murine IL-3 (Peprotech) or WEHI conditioned medium.
Growth Factor Mix
Mix IMDM, serum, BSA and growth factors
to create Growth Factor Mixes as listed below. 4 mL per assay (or
2 mL for BFU-E) will allow the cells to be plated in triplicate and can
be stored in aliquots at 4ºC o.n. or at -80ºC for
months. Multiply this volume x the number of experimental
groups and controls to be studied and by the number of cell doses that
will be plated in order to calculate the total amount of GF Mix
needed.
-
-
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
100 mg/mL10% 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 mGM-CSF
(100 ng/mL)
-
10% v/v -
-
-
huG-CSF
(100 ng/mL)
-
-
10% v/v -
-
Cell suspension
(See below)
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
Methylcellulose
in IMDM
-
-
-
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 5-FU treatment |
| Cells/mL x10(5) |
1-10 |
10-100 |
10-100 |
- Procedure (agar):
- 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.
- Procedure (methylcellulose):
- 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.
- Reading colonies:
-
Mix GM-CFC G-CFC BFU-E CFU-E 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
