How to Calculate Cell Concentration for Plating CFU Assays Following RBC Clearance with HetaSep™

When preparing small volume samples of fresh blood for CFU assays, red blood cells (RBCs) can be removed from your sample prior to plating via sedimentation. Such RBC-cleared samples may be subsequently used for colony-forming unit (CFU) assays to assess the proliferation and differentiation of hematopoietic stem and progenitor cells. This protocol describes how to calculate the cell concentration after a blood sample has been RBC-cleared with HetaSep™, and how to determine the volume of your RBC-cleared sample to achieve your desired plating density for a CFU assay.

Materials


Protocol

Please refer to the following definitions for notations used in the calculations below:

  • [ Sample ] = concentration of cells in the sample (i.e. cells/mL)
  • C = concentration
  • V = volume
  1. Perform a total nucleated cell count using 3% Acetic Acid with Methylene Blue to obtain the cell concentration of your starting sample. For more information, see this protocol for How to Count Cells with a Hemocytometer.
  2. Calculate the Dilution Factor by dividing the total diluted sample volume by the original sample volume:

    Example: If you are following this small-volume HetaSep™ protocol, start with a blood sample, and add 150 µL of Phosphate Buffered Saline (PBS) with 2% Fetal Bovine Serum (FBS) and 40 µL of HetaSep™. In this case, you would calculate the Dilution Factor as follows:

  3. Calculate the nucleated cell concentration of your sample after RBC clearance using HetaSep™ ([SampleRBC-cleared]). This is calculated by dividing the cell concentration of your starting sample by the Dilution Factor:

    Example: If the concentration of your starting sample was 5.0 x 106 cells/mL, and your Dilution Factor is 4.8, then the concentration of the sample after RBC clearance using HetaSep™ will be:

  4. You are now ready to prepare your sample for the CFU assay using MethoCult™ medium. As per standard protocol for CFU assays, prepare a 10X concentration of the desired plating density for dilution in the MethoCult™ medium. Use the RBC-cleared sample to prepare the 10X concentration, using the following formula:

    Example: If the required plating density for your sample is 1 x 104 cells per 35 mm culture dish, then you will need to prepare a 10X concentration of 1 x 105 cells/mL as follows:

    Therefore, you will need to add 96 µL of the RBC-cleared sample to 904 µL of IMDM with 2% FBS, to prepare the desired 10X concentrated sample for your CFU assay.
  • Document #PR00004
  • Version 1.0.0
  • Mar 2020


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