EasySep™小鼠TIL（CD45）正选试剂盒
Calculating Cell Concentration for Plating CFU Assays Following RBC Clearance with HetaSep™
Calculating Cell Concentration for Plating CFU Assays Following RBC Clearance with HetaSep™
When preparing small volume samples of fresh blood for CFU assays, we recommend using (Catalog #07806/07906) to remove the red blood cells from your sample prior to plating. A detailed protocol for using HetaSep™ on small volume samples of fresh blood is outlined in the Technical Bulletin: . Using this small volume HetaSep™ protocol, you only need to perform a cell count of your starting sample, and then you can determine the cell concentration of the RBC-cleared sample using the calculations below.
Step 1 - Perform a Total Nucleated Cell (TNC) count using (Catalog #07060) to obtain the cell concentration of your starting sample. For more information on cell counting please refer to this .
Step 2 - Calculate the Dilution Factor by dividing the total diluted sample volume by the original sample volume:
For this small volume HetaSep™ protocol, the blood sample is diluted using PBS with 2% FBS, and HetaSep™. According to this protocol, you should start with 50 µL of blood sample, and add (a) 150 µL of PBS with 2% FBS plus (b) 40 µL of HetaSep™. In this case, you would calculate the Dilution Factor as follows:
Step 3 - Calculate the nucleated cell concentration of your sample after RBC clearance using HetaSep™. This is calculated by dividing the cell concentration of your starting sample by the Dilution Factor:
For example, if the concentration of your starting sample was 5.0 x 106 cells per mL, and your Dilution Factor is 4.8, then the concentration of the sample after RBC clearance using HetaSep™ will be:
Step 4 - You are now ready to prepare your sample for the CFU assay using 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.
For 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 per mL. To prepare 1 mL of the 10X concentration, please use the following formula:
where, C10X = 1 x 105 cells per mL
V10X = 1 mL
C RBC-cleared sample = 1.04 x 106 cells per mL
V RBC-cleared sample = ?
In this case, the volume of RBC-cleared sample which is required to make 1 mL of the 10X concentration is:
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.
For more information about the CFU assay, please refer to the MethoCult™ Technical Manual.
For further assistance and information, please contact techsupport@stemcell.com.
Please refer the following definitions for notations used in the calculations above:
[ Sample ] = concentration of cells in the sample (i.e. cells/mL)
C = concentration
V = volume
Step 1 - Perform a Total Nucleated Cell (TNC) count using (Catalog #07060) to obtain the cell concentration of your starting sample. For more information on cell counting please refer to this .
Step 2 - Calculate the Dilution Factor by dividing the total diluted sample volume by the original sample volume:
For this small volume HetaSep™ protocol, the blood sample is diluted using PBS with 2% FBS, and HetaSep™. According to this protocol, you should start with 50 µL of blood sample, and add (a) 150 µL of PBS with 2% FBS plus (b) 40 µL of HetaSep™. In this case, you would calculate the Dilution Factor as follows:
Step 3 - Calculate the nucleated cell concentration of your sample after RBC clearance using HetaSep™. This is calculated by dividing the cell concentration of your starting sample by the Dilution Factor:
For example, if the concentration of your starting sample was 5.0 x 106 cells per mL, and your Dilution Factor is 4.8, then the concentration of the sample after RBC clearance using HetaSep™ will be:
Step 4 - You are now ready to prepare your sample for the CFU assay using 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.
For 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 per mL. To prepare 1 mL of the 10X concentration, please use the following formula:
where, C10X = 1 x 105 cells per mL
V10X = 1 mL
C RBC-cleared sample = 1.04 x 106 cells per mL
V RBC-cleared sample = ?
In this case, the volume of RBC-cleared sample which is required to make 1 mL of the 10X concentration is:
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.
For more information about the CFU assay, please refer to the MethoCult™ Technical Manual.
For further assistance and information, please contact techsupport@stemcell.com.
Please refer the following definitions for notations used in the calculations above:
[ Sample ] = concentration of cells in the sample (i.e. cells/mL)
C = concentration
V = volume
