Mouse Methylcellulose Complete Media for Pre-B Cells

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Catalog # Availability Size / Price Qty
HSC009
Product Details
Procedure
Citations (3)
FAQs
Reviews

Mouse Methylcellulose Complete Media for Pre-B Cells Summary

Kit Summary

For the growth and enumeration of mouse precursor B (pre-B) cells, optimized with IL-7.

Key Benefits

  • Optimized for enumeration of mouse pre-B cells
  • Includes premium quality recombinant IL-7 to promote proliferation of pre-B cell
  • Excellent optical clarity facilitates colony identification
  • High lot-to-lot consistency decreases variation
 

 

Why use R&D Systems Mouse Methylcellulose Complete Media for Pre-B Cells 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 for Pre-B Cells which contains Recombinant Mouse IL-7, a cytokine that promotes proliferation of pre- and pro-B cells of mouse origin. Pre-B cells are derived from progenitor B (pro-B) cells and mature into immature B cells in response to cytokines. In addition to their role in normal B cell development, pre-B and immature B cells are associated with certain leukemias.

R&D Systems Mouse Methylcellulose Complete Media for Pre-B cells:

  • Optical clarity facilitates colony identification.
  • High lot-to-lot consistency decreases variation.
  • Supports reproducible in vitro growth of pre-B progenitor cells of mouse origin.
  • Does not require addition of growth factors or cytokines.
  • Increased cloning efficiency and improved colony growth compared to agar.
 

 

Kit Contents
  • 100 mL of Mouse Methylcellulose Complete Media for Pre-B Cells.
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 20%
L-Glutamine 2 mM
2-Mercaptoethanol 5 x 10-5 M
Recombinant Mouse IL-17 10 ng/mL

Stability and Storage

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

Precaution

  • 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 mouse hematopoietic progenitors and is ineffective with human hematopoietic progenitors.
  • Derivation of mouse hematopoietic progenitors from different individual animals may cause results to vary.
 

 

Data Examples

View Larger Image

Mouse Pre-B Colony Formation. Mouse Methylcellulose Complete Media for Pre-B Cells (Catalog # HSC009) was used in the colony forming assay to support growth of pre-B progenitor cells of mouse origin. Both images show colony forming unit Pre-B colonies at 40X magnification. Each individual colony consists of a homogeneous population of tiny, round or oval shaped cells. CFU Pre-B colonies vary in size and morphology. Some colonies form a tight and compact spheroid while others are diffuse colonies with cells in the periphery.

 

 

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

Mouse 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
HSC006 Mouse Methylcellulose 90 mL N/A* Yes None
HSC011 StemXVivo® Methylcellulose
Concentrate
50 mL N/A* No None

Complete Mouse Methylcellulose Media

Catalog # Product Description Volume Colonies Selected for Contains Serum Cytokines Included
HSC007 Mouse Methylcellulose Complete Media 100 mL BFU-E
CFU-E
CFU-G
CFU-GEMM
CFU-GM
CFU-M
Yes Epo
IL-3
IL-6
SCF
HSC008 Mouse Methylcellulose
Complete Media Without
Epo
100 mL CFU-G
CFU-GM
CFU-M
Yes IL-3
IL-6
SCF
HSC009 Mouse Methylcellulose
Complete Media for
Pre-B Cells
100 mL CFU-Pre-B Yes IL-7

*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.

Product Datasheets

Preparation and Storage

Stability & 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.

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, Mouse Methylcellulose Complete Media for Pre-B Cells is used in the Colony Forming Cell Assay using the following procedure:

  • Prepare mouse bone marrow cells
  • Add cells to Mouse Methylcellulose Complete Media for Pre-B Cells
  • Plate and incubate cells
  • Identify and count colonies
 

 

Reagents Provided

Reagent supplied in the Mouse Methylcellulose Complete Media for Pre-B Cells (Catalog # HSC009):

  • 100 mL of Mouse Methylcellulose Complete Media for Pre-B Cells.
Contents Concentration
(when diluted to a final volume of 100 mL)
Methylcellulose (1500 cps) in
Iscove's Modified Dulbecco's Medium
1.3%
Fetal Bovine Serum 20%
L-Glutamine 2 mM
2-Mercaptoethanol 5 x 10-5 M
Recombinant Mouse IL-7 10 ng/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.

Pass a suspension of mouse bone marrow cells through a 70 µm nylon strainer

Remove the supernatant.

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

Remove the supernatant

Thaw aliquots of Mouse Methylcellulose Complete Media for Pre-B Cells 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 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/2% FBS 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 Mouse Methylcellulose Complete Media for Pre-B Cells. 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.

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

Citations for Mouse Methylcellulose Complete Media for Pre-B Cells

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

3 Citations: Showing 1 - 3
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  1. New therapeutic opportunities from dissecting the pre-B leukemia bone marrow microenvironment
    Authors: LC Cheung, J Tickner, AM Hughes, P Skut, M Howlett, B Foley, J Oommen, JE Wells, B He, S Singh, GA Chua, J Ford, CG Mullighan, RS Kotecha, UR Kees
    Leukemia, 2018;0(0):.  2018
  2. Precursor B Cells Increase in the Lung during Airway Allergic Inflammation: A Role for B Cell-Activating Factor
    PLoS ONE, 2016;11(8):e0161161.  2016
  3. Arsenite selectively inhibits mouse bone marrow lymphoid progenitor cell development in vivo and in vitro and suppresses humoral immunity in vivo.
    Authors: Ezeh, Peace C, Lauer, Fredine, MacKenzie, Debra, McClain, Shea, Liu, Ke Jian, Hudson, Laurie G, Gandolfi, A Jay, Burchiel, Scott W
    PLoS ONE, 2014;9(4):e93920.  2014

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