– UNM Comprehensive Cancer Center

M. Fero 11/20/2011

Materials:
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.

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.

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.

Heat inactivated FBS

Serum should be heat-inactivated to remove complement. This is important if the cells have been stained with antibodies and flow sorted by flow cytometry. Place a 50 mL serum aliquot in a 56C water bath for 30 min. and then cool. Spin the tube at 2500 RPM for 5 min to pellet insoluble proteins. Transfer the supernatant to a fresh 50 mL conical tube.

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/mL	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
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.

Incubation:
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

Tags: Fero-Lab-Protocols

Hematopoietic CFC Assays