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Human Methylcellulose Serum-Free Enriched Media Without Epo

R&D Systems, part of Bio-Techne | Catalog # HSC010SF

R&D Systems, part of Bio-Techne

Key Product Details

Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay.

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
 

 

Reagents Provided

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

 

Other Supplies Required

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

 

Procedure Overview

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.

Pass a suspension of mouse bone marrow cells through a 70 µm nylon strainer

Thaw aliquots of Human Methylcellulose Serum-Free Enriched Media Without Epo at room temperature for approximately 30 minutes.

Remove the supernatant

Resuspend mononuclear cells in 10 mL of IMDM.

Thaw aliquots of Methylcellulose Stock Solution at room temperature

Perform a cell count.

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.

Transfer the appropriate volume of cells plus a slight excess into a new 15 mL centrifuge tube

Remove the supernatant.

Resuspend the cells in IMDM to the desired stock cell number to generate a 10X stock concentration.

Remove the supernatant

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%

Combine the appropriate volume of 10X cell stock

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.

Vortex the samples vigorously

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.

Place two 35 mm plates into a 10 cm plate

Use an inverted microscope and a scoring grid to identify and count individual colonies.

Place two 35 mm plates into a 10 cm plate
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