Rat Methylcellulose Complete Media Without Epo

Catalog # Availability Size / Price Qty
HSC012
Product Details
Procedure
FAQs
Reviews

Rat Methylcellulose Complete Media Without Epo Summary

Kit Summary

Complete media for the differentiation and enumeration of rat colony-forming myeloid progenitors (CFU-GM) and for the identification of Epo-independent erythropoiesis.

Key Benefits

  • Suitable for identification of Epo-independent erythropoiesis
  • Excellent optical clarity facilitates colony identification
  • High lot-to-lot consistency decreases variation
  • Optimized for enumeration of colony-forming myeloid progenitors (CFU-GM)
 

 

Why use R&D Systems Rat 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 Rat Methylcellulose Complete Media Without Epo, which contains premium quality recombinant rat cytokines to support the optimal growth and enumeration of colony-forming myeloid progenitors (CFU-GM) of rat origin. The absence of Epo makes this media useful for the identification of Epo-independent stimulators of erythropoiesis.

R&D Systems Rat Methylcellulose Complete Media Without Epo:

  • Can be used to identify Epo-independent stimulators of erythropoiesis.
  • Includes premium quality rat recombinant cytokines to support growth of CFU-GM.
  • Optical clarity facilitates colony identification.
  • High lot-to-lot consistency decreases variation.
  • Does not require addition of serum or cytokines.
 

 

Kit Contents
  • 100 mL of Rat Methylcellulose Complete Media Without Epo.

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 Rat SCF 50 ng/mL
Recombinant Rat GM-CSF 10 ng/mL
Recombinant Rat IL-3 10 ng/mL

Stability and Storage

Rat Methylcellulose Complete Media Without Epo 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 rat hematopoietic progenitors and is ineffective with human hematopoietic progenitors.
  • Derivation of human hematopoietic progenitors from different individuals may cause results to vary.
 

 

Data Examples

View Larger Image

Rat Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. A single cell suspension of rat bone marrow cells was resuspended in Rat Methylcellulose Complete Media Without Epo (Catalog # HSC012) and used in the Colony Forming Cell (CFC) assay. Colonies were viewed under light microscope after an 8-12 day incubation. A. Colony forming unit-granulocyte, macrophage (CFU-GM) are progenitors that give rise to colonies containing a heterogeneous population of macrophages and granulocytes. B and C. Colony forming unit- macrophage (CFU-M) are clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages.

 

 

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

Rat Methylcellulose Stock and Base Media

Catalog # Product Description Volume Colonies Selected for Contains Serum Cytokines Included
HSC001 Methylcellulose Stock Solution 100 mL N/A* No None
HSC011 StemXVivo® Methylcellulose
Concentrate
50 mL N/A* No None

Complete Rat Methylcellulose Media

Catalog # Product Description Volume Colonies Selected for Contains Serum Cytokines Included
HSC012 Rat Methylcellulose
Complete Media Without Epo
100 mL CFU-G
CFU-GM
CFU-M
Yes GM-CSF
IL-3
SCF

*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.

Key Benefits

HSC Differentiation &
Lineage Marker Schematic


Hematopoietic Stem Cells & Lineage-specific Markers

View Interactive Pathway

The definitive hematopoietic system is made up of all adult blood cell types including megakaryocytes, erythrocytes, and cells of the myeloid and lymphoid lineages. All of these cells are derived from multipotent hematopoietic stem cells (HSCs) through a succession of precursors with progressively limited potential. Hematopoietic stem cells are tissue-specific stem cells that exhibit remarkable self-renewal capacity and are responsible for the life-long mainte­nance of the hematopoietic system. HSCs are rare cells that reside in adult bone marrow where hematopoiesis is continu­ously taking place. They can also be found in cord blood, fetal liver, adult spleen, and peripheral blood. R&D Systems offers several products for studying hematopoietic lineage cells including serum-free media, lineage deple­tion antibodies and kits, and reagents for performing colony forming cell (CFC) assays.

Specifications

Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
Rat

Product Datasheets

Background: Hematopoietic Stem Cells

HSC Differentiation &
Lineage Marker Schematic


Hematopoietic Stem Cells & Lineage-specific Markers

View Interactive Pathway

The definitive hematopoietic system is made up of all adult blood cell types including megakaryocytes, erythrocytes, and cells of the myeloid and lymphoid lineages. All of these cells are derived from multipotent hematopoietic stem cells (HSCs) through a succession of precursors with progressively limited potential. Hematopoietic stem cells are tissue-specific stem cells that exhibit remarkable self-renewal capacity and are responsible for the life-long mainte­nance of the hematopoietic system. HSCs are rare cells that reside in adult bone marrow where hematopoiesis is continu­ously taking place. They can also be found in cord blood, fetal liver, adult spleen, and peripheral blood. R&D Systems offers several products for studying hematopoietic lineage cells including serum-free media, lineage deple­tion antibodies and kits, and reagents for performing colony forming cell (CFC) assays.

Alternate Names
Hematopoietic Stem Cells

Assay Procedure

Refer to the product datasheet for complete product details.

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

  • Prepare rat bone marrow cells
  • Add cells and desired supplements to Rat Methylcellulose Complete Media Without Epo
  • Plate and incubate cells
  • Identify and count colonies
 

 

Reagents Provided

Reagent supplied in the Rat Methylcellulose Complete Media Without Epo (Catalog # HSC012):

  • 100 mL of Rat Methylcellulose Complete Media Without Epo.
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 Rat SCF 50 ng/mL
Recombinant Rat GM-CSF 10 ng/mL
Recombinant Rat IL-3 10 ng/mL

 

Other Supplies Required

Reagents

  • Cells derived from rat bone marrow, spleen, peripheral blood, or fetal liver.
  • 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

Prepare a suspension of rat 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.

Prepare a suspension of rat bone marrow cells through a 70 &icro;m nylon strainer

Remove the supernatant.

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

Remove the supernatant

Thaw aliquots of Rat Methylcellulose Complete Media Without Epo at room temperature for approximately 30 minutes.

Thaw aliquots of Rat Methylcellulose Complete Media Without Epo 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 8 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 Rat Methylcellulose Complete Media Without Epo. The final Methylcellulose concentration should be 1.3%.

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.

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

Reagents Provided

Other Reagents Provided

FAQs

No product specific FAQs exist for this product, however you may

View all Stem Cell Product FAQs

Reviews for Rat Methylcellulose Complete Media Without Epo

There are currently no reviews for this product. Be the first to review Rat Methylcellulose Complete Media Without Epo and earn rewards!

Have you used Rat Methylcellulose Complete Media Without Epo?

Submit a review and receive an Amazon gift card.

$25/€18/£15/$25CAN/¥75 Yuan/¥1250 Yen for a review with an image

$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image

Submit a Review