The Intelligent Flow Cytometric Cell Differentiation Assay


Buy HemaFlow™ Reagents


For decades, investigators have been glued to the methylcellulose colony-forming unit (CFU) assay, not because they want to count colonies, but because they have been told that they need to see differentiated and mature cells in the colonies so that they can identify them as belonging to a specific hematopoietic lineage.

HemaFlow™ now provides the intelligent alternative to that paradigm. By allowing cells to grow in suspension cultures for a shorter period of time and identifying and quantifying lineage-specific cells by flow cytometry using readily available fluorochrome-conjugated, antibody membrane biomarkers, the use of instrument-based, cell differentiation detection protocols make counting colonies obsolete.

  • Instrument-based to drastically reduce subjectivity.
  • Simple and fast assay setup in a 96-well plate (provided in the assay kit).
  • Short 4-5 day incubation for mouse cells and 5-7 day incubation for human cells.
  • Uses human or mouse CD markers or other mouse lineage-specific membrane markers to define specific differentiated cell populations, as opposed to counting and identifying colonies in a  methylcellulose CFU differentiation assay.
  • Use HemaFlo™ to replace the CFU assay for hematopoietic cellular therapy products.
  • Easy, rapid and accurate dispensing of HemaFlow™ Reagents using normal pipettes or liquid handlers.
  • Suspension cultures provide important cell interactions (not present using the CFU assay) that allow: (1) shorter lag time to initiate cell proliferation; (2) more rapid culture completion; (3) greater sensitivity.
  • HemaFlow™ can be adapted for high-throughput and high-content screening.
  • Setup as many replicates as necessary.
  • Use any flow cytometer available.
  • Add counting beads to better quantify results.
  • If necessary, initially verify results using either HALO®, HemoFLUOR™ or HemoLIGHT™ Research Assay Kits for same cell population being analyzed.

HemaFlow™ is available as low serum and serum-free formulations for:

  • Human
  • Mouse

HemaFlow™ can be used with the following fractionated cells or purified cell populations:

Human Mononuclear Cell (MNC) Fraction, CD34+ purified or CD133+ purified

  • Bone marrow
  • Umbilical cord blood
  • Peripheral blood (normal or mobilized).
Preferred Cell Systems™ recommends using Miltenyi Biotec magnetic bead separation technology for purifying CD34+ and CD133+ cells.


  • Bone marrow (cells can be fractionated to an MNC fraction and may also be purified)
  • Spleen (purified cell population may be necessary)
  • Fetal liver
  • Embryonic tissue
  • SC-GEMM 1: Primitive hematopoietic stem cell
  • P-BFU 1: Primitive erythropoietic progenitor cell
  • P-GM 1: Primitive granulocyte-macrophage progenitor cells 
  • P-Mk 1: Primitive megakaryopoietic progenitor cell

For background controls, use HemoGro™  medium alone.

HemaFlow™ is a replacement for the methylcellulose CFU assay using in cellular therapy processing laboratories for umbilical cord blood, bone marrow or mobilized peripheral blood.

Insread of counting and differentiating colonies based on identifying cell types within the colony, readily available CD biomarkers for different cell populations and lineages (see below) make the process fast, easy and more cost-effective.

  • Add 0.09mL of HemaFlow™ Reagent to wells in the 96-well plate (provided) and add 0.01mL of the cell suspension (50,000 cells/mL) and incubate for 5 - 7 days. HemaFlow™, like the CFU assay, detects cell differentiation capability and require cell growth to occur. 
  • Process the cells directly in the wells or remove cells to tubes and stain with required antibodies.
  • Use a flow cytometer with 4 or more fluorescent channels. 
  • Setup as many replicate wells as necessary. 
  • Combine HemaFlow™ with outher flow cytometric readouts. 
  • Quality control the instrument. 
  • Use counting beads to obtain an absolute cell number. 
  • Standardize the gating procedure for each cell population.
  • Training is simple and quick.
PLEASE NOTE: Neither HemaFlow™ nor the CFU assay should not be considered as potency assays. Neither assay measures cell proliferation, which is one of the requirments for a cell potency assay. For a cell potency assay, please see HALO® SC-IPS

The table below shows the CD antibodies used to detect different cell populations from different human and mouse HemaToxFlow™ Assay Kits. 


Product / Catalog Number Suggested Antibodies Cell Population Detected
HemaFlow™Assay Kit for Human Stem Cells*. HF(SF)-GEMM1-100H CD34
CD235a (GlyA)
Hematopoietic stem cells
Primitive hematopoietic stem cells
(Lineage-specific cells produced from the SC-GEMM 1 stem cell population can be detected using CD antibody panels in addition to CD34 and CD133. This will be similar to differentiating and counting different colonies in a CFU assay).
HemaFlow™ Assay Kit for Human Erythroid Cells. HF(SF)-BFU1-100H CD235a (GlyA) Erythroid cells
HemaFlow™ Assay Kit for Human Myelod Cells. HF(SF)-GM1-100H CD13
Myelod progenitor cells
HemaFlow™ Assay Kit for Human Megakaryocytes. HF(SF)-MK1-100H CD41
HemaFlo™ Assay Kit for Murine Stem Cells*. HF(SF)-GEMM1-100M CD34 Hematopoietic stem cells
HemaFlo™ Assay Kit for Murine Erythroid Cells. HF(SF)-BFU1-100M CD235a
Erythroid cells
HemaFlo™ Assay Kit for Murine Myeloid Cells. HF(SF)-GM1-100M Mac-1/CD11b
HemaFlo™ Assay Kit for Murine Megakaryocytic. HF(SF)-Mk1-100M CD41

Lineage-specific cells, produced by the stem cell reagent, can also be detected using panels of the suggest antibodies for either human or mouse. Cells in specific lineages, e.g. erythroid, myeloid or megakaryocytic can then be detected using gating procedures. However, it should be noted that detecting toxicity in different lineages using the HemaToxFlo™ Stem Cell Reagent may not produce the same results obtained with the HemaToxFlow™ Reagents for individual lineages.
(SF) Add "SF" to the catalog number for serum-free reagents.

The following QuickGuide shows the steps for using HemaFlow™

Method for performing HemaFlow™

  • HemaFlow™ Reagent. Please choose cell population and serum formulation.
  • Sterile, 96-well plate
  • Sterile, adhesive foil cover to maintain sterility of unused wells
Download the HemaToxFlow™ Manual
Certificate of Analysis (CoA) for HemaToxFlow™ are sent to the customer upon request 
Download the SDS Sheet