Can the CFU assay using Methycellulose based media be performed using frozen PBMCs instead of fresh PBMCs?

Name: Human Methylcellulose Serum-Free Base Media
Brand: R&D Systems
SKU: HSC002SF

Yes, the CFU assay can be performed using frozen PBMCs. The PBMCs can be frozen in DMEM containing 10% FBS and 10% DMSO.

Human Methylcellulose Serum-Free Base Media

Catalog # HSC002SF | R&D Systems, Inc. a Bio-Techne Brand

Citations (4)

Product Datasheet / COA / SDS

Key Product Details

Discontinued Product

HSC002SF has been discontinued. View all Hematopoietic Stem Cells products.

Setting the standard in quality research reagents

Summary for Human Methylcellulose Serum-Free Base Media

Kit Summary

Defined media for human hematopoietic stem cell growth and differentiation.

Key Benefits

Can be supplemented with user-defined cytokines and growth factors
Excellent optical clarity facilitates colony identification
High lot-to-lot consistency decreases variation

HSC002SF Data Examples
View Data Examples

Why use R&D Systems Human Methylcellulose Serum-Free Base 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. Additionally, inter-assay variation can be decreased by using defined media. R&D Systems offers Human Methylcellulose Serum-Free Base Media with superior optical clarity to support optimal colony growth, enumeration, and identification.

R&D Systems Human Methylcellulose Serum-Free Base Media:

Optical clarity facilitates colony identification.
High lot-to-lot consistency decreases variation.
Supports reproducible in vitro growth of hematopoietic stem and progenitor cells.
Can be supplemented with user-defined cytokines and growth factors.
Increased cloning efficiency and improved colony growth compared to agar.

Kit Contents

90 mL of Human Methylcellulose Serum-Free Base 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%
Human Transferrin	300 μg/mL
Bovine Serum Albumin	3.6%
Recombinant Human Insulin	16 μg/mL
L-Glutamine	2 mM
2-Mercaptoethanol	5 x 10^-5 M
Cholesterol	Trace

Stability and Storage

Human Methylcellulose Base Media 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.
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, and p24 antigen 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 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.

Data Examples

Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. A. 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.

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

Human 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
HSC002	Human Methylcellulose Base Media	90 mL	N/A*	Yes	None
HSC002SF	Human Methylcellulose Serum-Free Base Media	90 mL	N/A*	No	None
HSC011	StemXVivo^® Methylcellulose Concentrate	50 mL	N/A*	No	None

Complete Human Methylcellulose Media

Catalog #	Product Description	Volume	Colonies Selected for	Contains Serum	Cytokines Included
HSC003	Human Methylcellulose Complete Media	100 mL	BFU-E CFU-E CFU-G CFU-GEMM CFU-GM CFU-M	Yes	Epo GM-CSF IL-3 SCF
HSC004	Human Methylcellulose Complete Media without Epo	100 mL	CFU-G CFU-GM CFU-M	Yes	SCF GM-CSF IL-3
HSC005	Human Methylcellulose Enriched Media	100 mL	BFU-E CFU-E CFU-G CFU-GEMM CFU-GM CFU-M	Yes	Epo G-CSF GM-CSF IL-3 IL-6 SCF
HSC005SF	Human Methylcellulose Serum-Free Enriched Media	100 mL	BFU-E CFU-E CFU-G CFU-GEMM CFU-GM CFU-M	No	Epo G-CSF GM-CSF IL-3 IL-6 SCF
HSC010SF	Human Methylcellulose Serum-Free Enriched Media without Epo	100 mL	CFU-G CFU-GM CFU-M	No	G-CSF GM-CSF IL-3 IL-6 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.

HSC Differentiation &Lineage Marker Schematic 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.

Product Specifications for Human Methylcellulose Serum-Free Base Media

Species

Human

Scientific Data Examples for Human Methylcellulose Serum-Free Base Media

	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.

Preparation & Storage

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.

Assay Procedure

Refer to the product datasheet for complete product details.

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

Prepare human mononuclear cells
Add cells and desired supplements to Human Methylcellulose Base Media
Plate and incubate cells
Identify and count colonies

View Human Procedure

Reagents Provided

Reagents supplied in the Human Methylcellulose Serum-Free Base Media (Catalog # HSC002SF):

90 mL of Human Methylcellulose Serum-Free Base 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%
Human Transferrin	300 µg/mL
Bovine Serum Albumin	3.6%
Recombinant Human Insulin	16 µg/mL
L-Glutamine	2 mM
2-Mercaptoethanol	5 x 10^-5 M
Cholesterol	Trace

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.

Prepare mononuclear cells by Ficoll-Paque gradient centrifugation

Thaw aliquots of Human Methylcellulose Serum-Free Base Media at room temperature.

Thaw aliquots of Methylcellulose Stock Solution 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 5 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 Serum-Free Base 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 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.

Place two 35 mm plates into a 10 cm plate

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

Citations for Human Methylcellulose Serum-Free Base Media (4)

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Citations are publications that use Bio-Techne products. Selected citations for Human Methylcellulose Serum-Free Base Media include:

Species: Human
Sample Types: Whole Cells
Applications: Bioassay

I Antony-Deb et al. (2017-10-30), Pharmacological inhibition of the transcription factor PU.1 in leukemia J. Clin. Invest., 2017-10(0).
PMID: 29083320
Species: Human
Sample Types: Whole Cells
Applications: Bioassay

RF Stanley et al. (2017-02-23), A myeloid tumor suppressor role for NOL3 J. Exp. Med, 2017-00(0).
PMID: 28232469
Species: Human
Sample Types: Whole Cells

Pandolfi A et al. (2015-07-13), PAK1 is a therapeutic target in acute myeloid leukemia and myelodysplastic syndrome. Blood, 2015-0126(9):1118-27.
PMID: 26170031
Species: Human
Sample Types: Whole Cells

Barreyro L et al. (2012-06-21), Overexpression of IL-1 receptor accessory protein in stem and progenitor cells and outcome correlation in AML and MDS. Blood, 2012-0120(6):1290-8.
PMID: 22723552

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