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.
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
- 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.
Calculate the Dilution Factor by dividing the total diluted sample volume by the original sample volume:
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:
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: