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Human Methylcellulose Complete Media Without Epo

Key Product Details

Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay.
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HSC004

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, Human Methylcellulose Complete Media Without Epo is used in the Colony Forming Cell Assay using the following procedure:

  • Prepare human mononuclear cells
  • Add cells to Human Methylcellulose Complete Media Without Epo
  • Plate and incubate cells
  • Identify and count colonies
 

 

Reagents Provided

Reagents supplied in the Human Methylcellulose Complete Media (Catalog # HSC004):

  • 100 mL of Human Methylcellulose Complete 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%
Fetal Bovine Serum 25%
Bovine Serum Albumin 2%
L-Glutamine 2 mM
2-Mercaptoethanol 5 x 10-5 M
Recombinant Human SCF 50 ng/mL
Recombinant Human GM-CSF 10 ng/mL
Recombinant Human IL-3 10 ng/mL

 

Cell Resuspension Solution (15 mL)

Contents Concentration
Fetal Bovine Serum in
Iscove’s Modified Dulbecco’s Medium
50%

 

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 Methylcellose Complete Media Without Epo 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 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 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 Complete 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 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

  • What are the differences between in Methylcellulose Complete Media with EPO (Catalog # HSC003) and without EPO (Catalog # HSC004)?

    EPO is a potent cytokine used to stimulate differentiation of HSCs. EPO is needed for erythrocyte development. Media without EPO offers researchers the ability to expand and differentiate their HSCs and then to test supplemental drugs or compounds of interest.

  • 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.

  • Why does the Human, Mouse and Rat colony forming assay protocol (CFC assay protocol) recommed use of non-tissue culture treated petri dishes?

    The CFC assay promotes the growth of cells as colonies suspended in methylcellulose. However, if you use tissue culture treated dishes, the cells will also adhere and grow out on the bottom of the plate. Sometimes this appears as a round colony that is sticking and growing out on the edges (like an egg) and sometimes you can see patches of a monolayer. This makes it difficult to see the suspended colonies.

  • Burst Forming Unit-Erythroid (BFU-E ) colonies representing erythorid progenitors appear to be low in frequency.  Is there a strategy to count these colonies and visualize them?

    It is true that BFU-E colonies are low in frequency. To count and see good BFU-E colonies,  the CFC assay is set up at two cell densities.  For counting BFU-E colonies, a 10X cell concentration of  1.5-3x105 cells/mL  is used. For properly visualizing the BFU-E colonies,  an assay at half that cell density is used.

Product Documents

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