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Mouse Methylcellulose Complete Media

Catalog #: HSC007
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Mouse Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay.
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Mouse Methylcellulose Complete Media Summary

Kit Summary

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

Key Benefits

  • Does not require addition of serum or cytokines
  • Excellent optical clarity facilitates colony identification
  • High lot-to-lot consistency decreases variation
 

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.

 

Kit Contents
  • 100 mL of Mouse Methylcellulose Complete Media.

Mouse Methylcellulose Complete Media (100 mL)

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
Human Transferrin 200 μg/mL
Recombinant Human Insulin 10 μg/mL
Recombinant Human SCF 50 ng/mL
Recombinant Mouse IL-3 10 ng/mL
Recombinant Mouse IL-6 10 ng/mL
Recombinant Human Epo 5 IU/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.
 

 

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.

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
Mouse

Product Datasheets

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Scientific Data

Cell Morphology Mouse Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. View Larger

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

Prepare mononuclear cells by Ficoll-Paque gradient centrifugation

Thaw aliquots of Mouse Methylcellulose Complete Media 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. 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

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.

30 Citations: Showing 1 - 10
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  1. Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia
    Authors: Kovuru, N;Mochizuki-Kashio, M;Menna, T;Jeffrey, G;Hong, Y;Me Yoon, Y;Zhang, Z;Kurre, P;
    Nature communications  2024-02-29
  2. Preclinical Assessment of ADAM9-Responsive Mesoporous Silica Nanoparticles for the Treatment of Pancreatic Cancer
    Authors: Slapak, EJ;El Mandili, M;Brink, MST;Kros, A;Bijlsma, MF;Spek, CA;
    International journal of molecular sciences  2023-06-27
  3. Proof of Concept of the Radiosensitizing Effect of Gadolinium Oxide Nanoparticles in Cell Spheroids and a Tumor-Implanted Murine Model of Chondrosarcoma
    Authors: MT Aloy, J Sidi Boume, A Deville, D Kryza, A Gauthier, D Brichart-V, G Ollier, V La Padula, F Lux, O Tillement, C Rodriguez-, M Janier
    International Journal of Nanomedicine, 2022-12-23;17(0):6655-6673.  2022-12-23
  4. Effect of spermidine on radiation-induced long-term bone marrow cell injury
    Authors: B Guan, C Li, Y Yang, Y Lu, Y Sun, L Su, G Shi, L Bai, J Liu, A Meng
    International immunopharmacology, 2022-12-19;114(0):109557.  2022-12-19
  5. Influenza A virus infection instructs hematopoiesis to megakaryocyte-lineage output
    Authors: MGE Rommel, L Walz, F Fotopoulou, S Kohlscheen, F Schenk, C Miskey, L Botezatu, Y Krebs, IM Voelker, K Wittwer, T Holland-Le, Z Ivics, V von Messli, MAG Essers, MD Milsom, CK Pfaller, U Modlich
    Cell Reports, 2022-10-04;41(1):111447.  2022-10-04
  6. Pulmonary transplantation of alpha-1 antitrypsin (AAT)-transgenic macrophages provides a source of functional human AAT in vivo
    Authors: E Janosz, M Hetzel, H Spielmann, S Tumpara, C Rossdam, M Schwabbaue, D Kloos, C von Kaisen, A Schambach, FFR Buettner, S Janciauski, N Lachmann, T Moritz
    Gene Therapy, 2021-07-19;0(0):.  2021-07-19
  7. Chemotherapy-induced transposable elements activate MDA5 to enhance haematopoietic regeneration
    Authors: T Clapes, A Polyzou, P Prater, Sagar, A Morales-He, MG Ferrarini, N Kehrer, S Lefkopoulo, V Bergo, B Hummel, N Obier, D Maticzka, A Bridgeman, JS Herman, I Ilik, L Klaeylé, J Rehwinkel, S McKinney-F, R Backofen, A Akhtar, N Cabezas-Wa, R Sawarkar, R Rebollo, D Grün, E Trompouki
    Nature Cell Biology, 2021-07-12;23(7):704-717.  2021-07-12
  8. &alpha-Tocopherol Attenuates Oxidative Phosphorylation of CD34+ Cells, Enhances Their G0 Phase Fraction and Promotes Hematopoietic Stem and Primitive Progenitor Cell Maintenance
    Authors: L Rodriguez, P Duchez, N Touya, C Debeissat, AV Guitart, JM Pasquet, M Vlaski-Laf, P Brunet de, Z Ivanovic
    Biomolecules, 2021-04-10;11(4):.  2021-04-10
  9. A Transgenic Mouse Model of Pacak?Zhuang Syndrome with An Epas1 Gain-of-Function Mutation
    Authors: H Wang, J Cui, C Yang, JS Rosenblum, Q Zhang, Q Song, Y Pang, F Fang, M Sun, P Dmitriev, MR Gilbert, G Eisenhofer, K Pacak, Z Zhuang
    Cancers (Basel), 2019-05-14;11(5):.  2019-05-14
  10. Targeting VLA4 integrin and CXCR2 mobilizes serially repopulating hematopoietic stem cells
    Authors: D Karpova, MP Rettig, J Ritchey, D Cancilla, S Christ, L Gehrs, E Chendamara, MO Evbuomwan, M Holt, J Zhang, G Abou-Ezzi, H Celik, E Wiercinska, W Yang, F Gao, LG Eissenberg, RF Heier, SD Arnett, MJ Meyers, MJ Prinsen, DW Griggs, A Trumpp, PG Ruminski, DM Morrow, HB Bonig, DC Link, JF DiPersio
    J. Clin. Invest., 2019-05-14;129(7):2745-2759.  2019-05-14
  11. Bone marrow dendritic cells regulate hematopoietic stem/progenitor cell trafficking
    Authors: J Zhang, T Supakornde, JR Krambs, M Rao, G Abou-Ezzi, RY Ye, S Li, K Trinkaus, DC Link
    J. Clin. Invest., 2019-04-30;129(7):2920-2931.  2019-04-30
  12. miR-9 upregulation leads to inhibition of erythropoiesis by repressing FoxO3
    Authors: Y Zhang, L Li, C Yu, V Senyuk, F Li, JG Quigley, T Zhu, Z Qian
    Sci Rep, 2018-04-25;8(1):6519.  2018-04-25
  13. Pharmacological inhibition of the transcription factor PU.1 in leukemia
    Authors: I Antony-Deb, A Paul, J Leite, K Mitchell, HM Kim, LA Carvajal, TI Todorova, K Huang, A Kumar, AA Farahat, B Bartholdy, SR Narayanaga, J Chen, A Ambesi-Imp, AA Ferrando, I Mantzaris, E Gavathioti, A Verma, B Will, DW Boykin, WD Wilson, GM Poon, U Steidl
    J. Clin. Invest., 2017-10-30;0(0):.  2017-10-30
  14. A myeloid tumor suppressor role for NOL3
    Authors: RF Stanley, RT Piszczatow, B Bartholdy, K Mitchell, WM McKimpson, S Narayanaga, D Walter, TI Todorova, C Hirsch, H Makishima, B Will, C McMahon, K Gritsman, JP Maciejewsk, RN Kitsis, U Steidl
    J. Exp. Med, 2017-02-23;0(0):.  2017-02-23
  15. Ras oncogene-independent activation of RALB signaling is a targetable mechanism of escape from NRAS(V12) oncogene addiction in acute myeloid leukemia
    Oncogene, 2016-12-19;0(0):.  2016-12-19
  16. Paradoxical roles of elongation factor-2 kinase in stem cell survival
    J Biol Chem, 2016-07-27;0(0):.  2016-07-27
  17. Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency
    Stem Cell Reports, 2016-07-21;0(0):.  2016-07-21
  18. Monocyte/macrophage lineage commitment and distribution are affected by the lack of regulatory T cells in scurfy mice
    Eur J Immunol, 2016-05-27;0(0):.  2016-05-27
  19. Chemoprotection of murine hematopoietic cells by combined gene transfer of cytidine deaminase (CDD) and multidrug resistance 1 gene (MDR1).
    Authors: Brennig S, Lachmann N, Buchegger T, Hetzel M, Schambach A, Moritz T
    J Exp Clin Cancer Res, 2015-12-12;34(0):148.  2015-12-12
  20. The Corepressor Rcor1 Is Essential for Normal Myeloerythroid Lineage Differentiation.
    Authors: Yao H, Goldman D, Fan G, Mandel G, Fleming W
    Stem Cells, 2015-07-14;33(11):3304-14.  2015-07-14
  21. The transcription factor Foxm1 is essential for the quiescence and maintenance of hematopoietic stem cells.
    Authors: Hou Y, Li W, Sheng Y, Li L, Huang Y, Zhang Z, Zhu T, Peace D, Quigley J, Wu W, Zhao Y, Qian Z
    Nat Immunol, 2015-06-29;16(8):810-8.  2015-06-29
  22. Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes.
    Authors: Huan J, Hornick N, Goloviznina N, Kamimae-Lanning A, David L, Wilmarth P, Mori T, Chevillet J, Narla A, Roberts C, Loriaux M, Chang B, Kurre P
    Leukemia, 2015-06-25;29(12):2285-95.  2015-06-25
  23. Sensitivity and dose dependency of radiation-induced injury in hematopoietic stem/progenitor cells in mice.
    Authors: Guo C, Luo L, Urata Y, Goto S, Huang W, Takamura S, Hayashi F, Doi H, Kitajima Y, Ono Y, Ogi T, Li T
    Sci Rep, 2015-01-27;5(0):8055.  2015-01-27
  24. Corepressor Rcor1 is essential for murine erythropoiesis.
    Authors: Yao H, Goldman D, Nechiporuk T, Kawane S, McWeeney S, Tyner J, Fan G, Kerenyi M, Orkin S, Fleming W, Mandel G
    Blood, 2014-03-20;123(20):3175-84.  2014-03-20
  25. Promoter and lineage independent anti-silencing activity of the A2 ubiquitous chromatin opening element for optimized human pluripotent stem cell-based gene therapy.
    Authors: Ackermann M, Lachmann N, Hartung S, Eggenschwiler R, Pfaff N, Happle C, Mucci A, Gohring G, Niemann H, Hansen G, Schambach A, Cantz T, Zweigerdt R, Moritz T
    Biomaterials, 2013-11-26;35(5):1531-42.  2013-11-26
  26. Nicaraven attenuates radiation-induced injury in hematopoietic stem/progenitorcells in mice.
    Authors: Kawakatsu M, Urata Y, Imai R, Goto S, Ono Y, Nishida N, Li T
    PLoS ONE, 2013-03-29;8(3):e60023.  2013-03-29
  27. Placental extract protects bone marrow-derived stem/progenitor cells against radiation injury through anti-inflammatory activity.
    Authors: Kawakatsu M, Urata Y, Goto S, Ono Y, Li T
    J Radiat Res, 2012-11-14;54(2):268-76.  2012-11-14
  28. Cited2 gene controls pluripotency and cardiomyocyte differentiation of murine embryonic stem cells through Oct4 gene.
    Authors: Li Q, Ramirez-Bergeron D, Dunwoodie S, Yang Y
    J Biol Chem, 2012-07-03;287(34):29088-100.  2012-07-03
  29. The effects of low-dose ionizing radiation in the activated rat basophilic leukemia (RBL-2H3) mast cells.
    Authors: Joo H, Nam S, Yang K, Kim C, Jin Y, Kim J
    J Biol Chem, 2012-06-14;287(33):27789-95.  2012-06-14
  30. Hyperproliferation, cancer, and inflammation in mice expressing a Delta133p53-like isoform.
    Authors: Slatter TL, Hung N, Campbell H, Rubio C, Mehta R, Renshaw P, Williams G, Wilson M, Engelmann A, Jeffs A, Royds JA, Baird MA, Braithwaite AW
    Blood, 2011-03-16;117(19):5166-77.  2011-03-16

FAQs

  1. 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.

  2. 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.

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Mouse Methylcellulose Complete Media
By Leslie Priddy on 04/04/2018