Mouse Methylcellulose Complete Media

For the differentiation and enumeration of murine hematopoietic stem cells, optimized with premium quality cytokines.

Why use R&D Systems Mouse Methylcellulose Complete Media for Colony Forming Cell Assays?

Colony forming cell (CFC) assays, which are used to enumerate and quantify multi-potent and single lineage hematopoietic progenitors, can be time consuming and laborious.

Successful growth and enumeration of cell colonies is dependent on factors such as accurate cell counts, the presence of growth factors and/or cytokines, adequate humidity, and the use of high quality media. R&D Systems offers Mouse Methylcellulose Complete Media, which contains premium quality recombinant growth factors and cytokines, which support optimal colony growth and enumeration. The Mouse Methylcellulose Complete Media is specially formulated and has been optimized for CFC assays to identify burst-forming erythroid progenitors (BFU-E), colony-forming myeloid progenitors (CFU-GM), and the multi-potential progenitors (CFU-GEMM) of mouse origin.

R&D Systems Mouse Methylcellulose Complete Media:

• Supplemented with premium quality recombinant proteins.
• Optical clarity facilitates colony identification.
• High lot-to-lot consistency decreases variation.
• Supports reproducible in vitro growth of hematopoietic stem and progenitor cells.
• Does not require addition of serum or cytokines.
• Increased cloning efficiency and improved colony growth compared to agar.

• 100 mL of Mouse Methylcellulose Complete Media.

Mouse Methylcellulose Complete Media (100 mL)

Stability and Storage

Mouse Methylcellulose Complete Media should be stored at ≤-20 °C upon receipt. Storage at 2 °C to 8 °C is not recommended.

Precautions

• The acute and chronic effects of overexposure to this media are unknown. Safe laboratory procedures should be followed and protective clothing should be worn when handling this media.
• The human Transferrin used in this product was derived from human plasma, which has been tested and found negative for HIV-1/2 antibodies, Hepatitis B surface antigen, Hepatitis C antibody, Syphilis, ALT Test and NAT-PCR (HAV, HIV, HBC, HCV, and Parovirus B19) by FDA approved methods. Handle as if capable of transmitting infection, and dispose of according to applicable regulations

Limitations

• The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
• The reagent should not be used beyond the expiration date indicated on the label.
• Derivation of mouse hematopoietic progenitors from different individual animals may cause results to vary.
• The media is optimized to assay mouse hematopoietic progenitors and is ineffective with human hematopoietic progenitors.

Mouse Methylcellulose Stock and Base Media

Complete Mouse Methylcellulose Media

*Base media and stock solutions do not contain cytokines and will not support colony growth unless conditioned media, cytokines, or other culture supplements are added.

Scientific Data Examples for Mouse Methylcellulose Complete Media

Mouse Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. Burst forming unit-erythroid (BFU-E) colonies are defined as clusters with a minimal of 30 cells that can be seen from day 7 onward. Each individual cluster consisted of tiny, irregular shaped cells that may appear fused together. Each cluster normally contains 5 - 8 cells, and the size of the cluster is similar to that of a single macrophage. The cluster may vary in sizes and color. A large BFU-E is usually bright red and is differentiable even without the use of a microscope. Smaller BFU-E may not appear red in color but is distinguishable based on the morphology.B. Colony forming unit-macrophage (CFU-M; left) are clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages. Colony forming unit-granulocyte (CFU-G; right) are clonogenic progenitors of granulocytes that give rise to a homogeneous population of eosinophils, basophils, or neutrophils.C. Colony forming unit-granulocyte, macrophage (CFU-GM) are progenitors that give rise to colonies containing a heterogeneous population of macrophages and granulocytes. The morphology is similar to the CFU-M and CFU-G descriptions.D.Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) are multi-lineage progenitors that give rise to the lineage of erythroid, granulocytes, macrophages, and megakaryocytes as the name indicates. It can be identified as reddish colored cells (erythroid) mixed with colorless cells (granulocytes, macrophages, and megakaryocytes) in a single colony. This progenitor is typically the largest colony on the culture dish; occasionally CFU-GM may attain a size comparable or larger than that of CFU-GEMM.

Assay Procedure

Refer to the product datasheet for complete product details.

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

• Prepare mouse bone marrow cells
• Add cells to Mouse Methylcellulose Complete Media
• Plate and incubate cells
• Identify and count colonies

View Graphic Procedure

 

 

Reagents Provided

Reagent supplied in the Mouse Methylcellulose Complete Media (Catalog # HSC007):

• 100 mL of Mouse Methylcellulose Complete Media.

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	15%
Bovine Serum Albumin	2%
L-Glutamine	2 mM
2-Mercaptoethanol	5 x 10-5 M
Recombinant Human Insulin	10 µg/mL
Human Transferrin	200 µg/mL
Recombinant Human SCF	50 ng/mL
Recombinant Human IL-3	10 ng/mL
Recombinant Human IL-6	10 ng/mL
Recombinant Human Epo	5 IU/mL

 

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

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.

Remove the supernatant.

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

Thaw aliquots of Mouse Methylcellulose Complete Media at room temperature for approximately 30 minutes.

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 8 minutes.

Remove the supernatant.

Resuspend the cells in IMDM/2% FBS 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 Mouse Methylcellulose Complete Media. 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 8-12 days.

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