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Methylcellulose Stock Solution

Key Product Details

Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay.
(4)
Mouse Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. 
Tips to Identify CFU-GEMM Colonies in the Human Colony Forming Unit Assay.
Methylcellulose Stock Solution (Catalog # HSC001)
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HSC001

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, Methylcellulose Stock Solution is used in the Colony Forming Cell Assay using the following procedure:

  • Prepare human mononuclear cells or mouse bone marrow cells
  • Add cells and desired supplements to Methylcellulose Stock Solution
  • Plate and incubate cells
  • Identify and count colonies
 

 

Reagents Provided

Reagent supplied in the Methylcellulose Stock Solution (Catalog # HSC001):

  • 100 mL of 3% Methylcellulose in Iscove’s Modified Dulbecco’s Medium.
Contents Concentration
Methylcellulose (1500 cps) in
Iscove’s Modified Dulbecco's Medium
3.0%

 

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

 

Other Supplies Required

Reagents

  • Cells derived from mouse bone marrow, spleen, peripheral blood, or fetal liver. Mice are routinely used between 6 - 12 weeks.
  • Iscove's Modified Dulbecco’s Media (IMDM)
  • Fetal Bovine Serum
  • IMDM/2% Fetal Bovine Serum
  • (Optional) Flow Cytometry Mouse Lyse Buffer (Catalog # FC003)

Materials

  • 100 mm culture plates
  • 35 mm culture plates
  • 15 mL centrifuge tubes
  • 10 mL syringes
  • 3 mL syringes
  • 5 mL vials
  • 16 gauge 1½ inch needle
  • 14 gauge laboratory pipetting needle
  • Serological pipettes
  • Pipettes and pipette tips

Equipment

  • 37 °C and CO2 humidified incubator
  • Centrifuge
  • Vortex mixer
  • Hemocytometer
  • Inverted Microscope

 

Procedure Overview

Procedure for the Human Colony Forming Cell Assay

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 Methylcellulose Stock Solution 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 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 Methylcellulose Stock Solution. 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.

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

 

Procedure for the Mouse Colony Forming Cell Assay

Pass a suspension of mouse bone marrow cells through a 70 μm nylon strainer to remove clumps and debris.

Remove red blood cells if necessary.

Wash the cells with IMDM/2% FBS by centrifugation at 300 x g for 8 minutes and pool the cells.

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

Remove the supernatant.

Resuspend the cells in 10 mL of IMDM/2% FBS.

Remove the supernatant

Thaw aliquots of Methylcellulose Stock Solution at room temperature for approximately 30 minutes.

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 Methylcellulose Stock Solution. 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 8-12 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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    We use to make stem cell spheres using methylcellulose.

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FAQs

  • What is the difference between Methylcellulose Stock (Catalog # HSC001) and Base Media (Catalog # HSC002)?

    The Methylcellulose Stock (Catalog # HSC001) contains methylcellulose in Iscove's Modified Dulbecco's Medium. The Base Media (Catalog # HSC002) contains methylcellulose in Iscove's Modified Dulbecco's Medium along with serum, albumin, L-glutamine, and 2-mercaptoethanol. The additional components of HSC002 are needed to sustain the viability and growth of HSCs. The advantage to using HSC002 is that R&D Systems has already evaluated the serum, albumin, L-glutamine, and 2-mercaptoethanol in-house to ensure lot-to-lot consistency, eliminating the need for the researcher to evaluate these components individually.

  • 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.

  • Is lot-specific information on viscosity available for Methyl Cellulose Stock Solution (Catalog #  HSC001)?

    Methyl cellulose Stock Solution is prepared using Methyl cellulose with an estimated viscosity of 1500 CPS.  Because of our stringent quality control assays, we don’t expect large variations in viscosity from lot to lot.

  • Why is there color difference observed sometimes between two bottles of Methylcellulose Stock Solution (Catalog # HSC001)?

    The difference in color can be because of dry ice added to the shipment. Dry ice can cause minor pH changes and slight changes in color, but this will not affect performance.

  • What is the molecular weight of Methylcellulose used in Catalog # HSC001? 

    The Methylcellulose in Catalog # HSC001 has a molecular weight range of 208272 - 219639 g/mol.

  • 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.

  •  How many freeze-thaws cycles can Methylcellulose stock solution, Catalog # HSC001, undergo? 

    We recommend keeping freeze-thaw cycles to a minimum. In our experience, two freeze-thaw cycles of the material received is acceptable, but after that, the viscosity of the product may be affected. 

Product Documents

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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