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Human Methylcellulose Serum-Free Base Media

R&D Systems, Inc. a Bio-Techne Brand

Key Product Details

Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. 
Catalog #
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HSC002SF
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Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, Human Methylcellulose Base Media is used in the Colony Forming Cell Assay using the following procedure:

  • Prepare human mononuclear cells
  • Add cells and desired supplements to Human Methylcellulose Base Media
  • Plate and incubate cells
  • Identify and count colonies
 

 

Reagents Provided

Reagents supplied in the Human Methylcellulose Serum-Free Base Media (Catalog # HSC002SF):

  • 90 mL of Human Methylcellulose Serum-Free Base Media and 15 mL of Cell Resuspension Solution.
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
Bovine Serum Albumin 3.6%
Recombinant Human Insulin 16 µg/mL
L-Glutamine 2 mM
2-Mercaptoethanol 5 x 10-5 M
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.

Prepare mononuclear cells by Ficoll-Paque gradient centrifugation

Thaw aliquots of Human Methylcellulose Serum-Free Base Media at room temperature.

Thaw aliquots of Methylcellulose Stock Solution at room temperature

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 5 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 Cell Resuspension Solution 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 Base Media. 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 by gently rotating the plate.

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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FAQs for Human Methylcellulose Serum-Free Base Media

  • Can the CFU assay using Methycellulose based media be performed using frozen PBMCs instead of fresh PBMCs?

    Yes, the CFU assay can be performed using frozen PBMCs.  The PBMCs can be frozen in DMEM containing 10% FBS and 10% DMSO.

Product Documents for Human Methylcellulose Serum-Free Base Media

Certificate of Analysis

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