Human Methylcellulose Complete Media Without Epo

Kit Summary

For the differentiation and enumeration of granulocyte-macrophage progenitor cells and for detection of Epo-independent erythropoiesis, optimized with premium quality cytokines.

Why use R&D Systems Human Methylcellulose Complete Media Without Epo 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 Human Methylcellulose Complete Media Without Epo, which contains all of the growth factors and cytokines needed to support optimal growth and enumeration of colony-forming myeloid progenitors. The absence of Epo makes this media useful for the identification of Epo-independent stimulators of erythropoiesis. Human Methylcellulose Complete Media Without Epo is specially formulated and has been optimized for CFC assays using colony-forming myeloid progenitors (CFU-GM, CFU-G, CFU-M) of human origin. This product can also be used in the long-term culture-initiating cell (LTC-IC) assay.

R&D Systems Human Methylcellulose Complete Media without EPO:

• Can be used to identify Epo-independent stimulators of erythropoiesis.
• Optical clarity facilitates colony identification.
• High lot-to-lot consistency decreases variation.
• Supports reproducible in vitro growth of myeloid progenitor cells.
• Does not require addition of growth factors or cytokines.
• Increased cloning efficiency and improved colony growth compared to agar.

• 100 mL of Human Methylcellulose Complete Media Without Epo and 15 mL Cell Resuspension Solution.

Cell Resuspension Solution (15 mL)

Stability and Storage

Human Methylcellulose Complete Media Without Epo and the Cell Resuspension Solution 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.

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 vial labels.
• The media is optimized to assay human hematopoietic progenitors and is ineffective with mouse hematopoietic progenitors.
• Derivation of human hematopoietic progenitors from different individuals may cause results to vary.

Guide to Choosing Media for the Colony Forming Cell (CFC) Assay

Human Methylcellulose Stock and Base Media

Complete Human 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 Human Methylcellulose Complete Media Without Epo

Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. Colony forming unit-erythroid (CFU-E) are clonogenic progenitors that produce only one or two clusters with each cluster containing from 8 to approximately 100 hemoglobinized erythroblasts. It represents the more mature erythroid progenitors that have less proliferative capacity.B. Colony forming unit-granulocyte (CFU-G) 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. Burst forming unit-erythroid (BFU-E) colonies can be described as small (3 to 8 clusters), intermediate (9 to 16 clusters), or large (more than 16 clusters) according to the number of clusters present. These are primitive erythroid progenitors that have high proliferative capacity.E. Colony forming unit-macrophage (CFU-M) are clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages.F. Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) are multi-lineage progenitors that give rise to erythroid, granulocyte, macrophage and megakaryocyte lineages, as the name indicates.

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

View Human Procedure

 

 

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.

Thaw aliquots of Human Methylcellose Complete Media Without Epo at room temperature.

Resuspend mononuclear cells in 10 mL of IMDM.

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.

Remove the supernatant.

Resuspend the cells in Cell Resuspension Solution 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 Human Methylcellulose Complete Media Without Epo. 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 by gently rotating the plate.

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.

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