Human Methylcellulose Serum-Free Enriched Media Without Epo
R&D Systems, part of Bio-Techne | Catalog # HSC010SF
Key Product Details
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, Human Methylcellulose Serum-Free Enriched Media Without Epo is used in the Colony Forming Cell Assay using the following procedure:
- Prepare mouse bone marrow cells
- Add cells to Human Methylcellulose Serum-Free Enriched Media Without Epo
- Plate and incubate cells
- Identify and count colonies
Reagent supplied in the Human Methylcellulose Serum-Free Enriched Media Without Epo (Catalog # HSC010SF):
- 100 mL of Human Methylcellulose Serum-Free Enriched Media without Epo.
Contents | Concentration (when diluted to a final volume of 100 mL) |
Methylcellulose (1500 cps) in Iscove’s Modified Dulbecco’s Medium | 1.4% |
Human Transferrin | 300 µg/mL |
Fetal Bovine Serum | 3.6% |
Bovine Serum Albumin | 3.6% |
L-Glutamine | 2 mM |
2-Mercaptoethanol | 5 x 10-5 M |
Recombinant Human SCF | 50 ng/mL |
Human GM-CSF | 20 ng/mL |
Recombinant Human G-CSF | 20 ng/mL |
Recombinant Human IL-3 | 10 ng/mL |
Recombinant Human IL-6 | 10 ng/mL |
Cholesterol | trace |
Reagents
- Cells derived from bone marrow, blood, or enriched CD34+ cells
- Iscove’s Modified Dulbecco’s Media (IMDM)
- Ca2+/Mg2+-Free Hank's Balanced Salt Solution (HBSS)
- Ficoll-Paque™ PLUS (GE Healthcare) or equivalent
Materials
- 100 mm culture plates
- 35 mm culture plates
- 15 mL centrifuge tubes
- 50 mL centrifuge tubes
- 10 mL syringes
- 3 mL syringes
- 5 mL vials
- 16 gauge 1½ inch needle
- 14 gauge laboratory pipetting needle
- Heparinized syringes or Vacutainers®
- Serological pipettes
- Pipettes and pipette tips
Equipment
- 37 °C and CO2 humidified incubator
- Centrifuge
- Vortex mixer
- Hemocytometer
- Inverted Microscope
Prepare mononuclear cells by Ficoll-Paque gradient centrifugation.
Wash the cells two times with HBSS and pool the cells.
Centrifuge the cells at 400 x g for 10 minutes.
Thaw aliquots of Human Methylcellulose Serum-Free Enriched Media Without Epo at room temperature for approximately 30 minutes.
Resuspend mononuclear cells in 10 mL of IMDM.
Perform a cell count.
Transfer the appropriate volume of cells (plus a slight excess) into a new 15 mL centrifuge tube.
Centrifuge at 300 x g for 10 minutes.
Remove the supernatant.
Resuspend the cells in IMDM to the desired stock cell number to generate a 10X stock concentration.
Combine the appropriate volume of 10X cell stock with the desired cell culture supplements/cytokines, and Human Methylcellulose Serum-Free Enriched Media Without Epo. The final Methylcellulose concentration should be 1.27%
Vortex the samples vigorously.
Wait approximately 20 minutes to allow air bubbles to escape.
Add 1.1 mL of the cell mixture to a 35 mm culture plate using a 3 mL syringe and a 16 gauge needle.
Spread the media evenly.
Place two 35 mm plates into a 10 cm plate.
Add one uncovered 35 mm plate that contains 3-4 mL of sterile water.
Cover the 10 cm plate and place it in a 37 °C and 5% CO2 incubator.
Incubate the cells for 14-16 days.
Use an inverted microscope and a scoring grid to identify and count individual colonies.
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