WELCOME TO ASYMMETREX’S KINETIC STEM CELL (KSC) COUNTING PAGE!
If you study, produce, supply, or treat with tissue stem cells, like hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), imagine what it means for you to be able to quantify their numbers accurately for the first time without confounding with lineage-committed progenitor cells – in your experiments, in your biomanufacturing process, in your expansion bioreactors, or in your treatments, as the case may be. Just imagine what that will mean for your research progress, your manufacturing efficiency, your customer satisfaction, or the effectiveness of your stem cell and gene therapy treatments. Just imagine that for a moment…
Then, stop imagining and begin realizing these advances by taking advantage of the kinetic stem cell (KSC) counting technology available from Asymmetrex®.
Calculators I-III below provide some useful basic parameters for general mammalian in vitro cell culture. We would love to know what you are using the calculators for! How about letting us know with this optional survey.
Now, if you are using Calculators I-III for cell cultures that contain tissue stem cells, then you may be only one more calculator away from knowing how many stem cells are in your cultures. Because Calculators IV-VIII below provide free access to the KSC counting algorithms that Asymmetrex® is now debuting for rapid counting of 5 different preparations containing tissue stem cells. The free rapid-counting calculators require only routine 72-hour cell culture count data for use. Read more about them in two recent research reports published in 2022 and 2023 and start using them now below.
If you are already generating the basic tissue culture cell counts for Calculators I-III, then you are ready for Asymmetrex’s new generation of online calculators for rapid counting of tissue stem cells.
GET STARTED USING THEM TO IMPROVE YOUR TISSUE STEM CELL RESEARCH AND APPLICATIONS TODAY!
GENERAL CELL COUNTERS
I. Population doubling time (PDT)
The population doubling time of a culture is calculated by the following equation:
PDT in hours = ln2 * (hours of culture) / ln(fold change in cell number)
(Note: PDT is an estimate of the cell cycle time of the dividing cells in a culture only when the dividing fraction of new cells approaches 100% and the cell death rate is insignificant.)
To Calculate PDT:
- t1, time in hours when culture started (usually 0)
- t2, time in hours when culture period ended
- N1, number of cells in culture at time = t1
- N2, number of cells in culture at time = t2
| t1 | |
| t2 | |
| N1 | |
| N2 |
Result:
II. Cell Population Doublings
The number of cell population doublings during a period of culture is calculated by the following equation:
Number of cell doublings = ln(fold increase in cell number)/ln2
To calculate Cell Population Doublings:
- N1, Number of initial cells
- N2, Number of cells at the end of the period of culture
| N1 | |
| N2 |
Result:
III. Cumulative Population Doublings (CPD)
A CPD data graph can be generated by calculating the number of cell doublings for a series of consecutive culture intervals and summing the values sequentially.
Initial Cell Number:
Result:
TISSUE STEM CELL COUNTERS
IV. Asymmetrex® Rabbit Algorithm For Human Umbilical Cord Blood CD34+ HSCs
This calculator allows determination of the tissue stem cell-specific fraction (SCF) of human HSCs in CD34+-selected human umbilical cord blood when cultured in the following commercial cell culture medium:Stemcell Technologies, StemSpanTM SFEM II (Cat#09655); StemSpanTM CD34+ Expansion Supplement (Cat#02691); pen/strep.
Culture and counting procedures
1. In a 24-well plate, initiate evaluation cultures with ideally 50,000 to 100,000 total viable cells per 2.25 mLs of the culture medium specified above. Fewer cells can be used, but more cells should not be used. Recommend preparing 6 replicate wells.
2. Use 3 of the cultures to determine the mean total cell count 4-5 hours after plating. This analysis sets time = 0 hours.
3. Approximately 72 hours later, determine the mean total cell count in the remaining 3 cultures, time = "72 hours." This period should not be less than 66 hours and not more than 78 hours (i.e., range in fractional days from 2.75 to 3.25).
Note: Accurate tissue stem cell counting requires an increase in total cell number during the period of this analysis.
Calculator Procedure
1. Enter, d, the number of days of serial culture from the first cryopreservation of the initial isolated primary tissue cell preparation (e.g., 0, 3.0, 6.0). Entry of fractional days is recommended for increased accuracy.
Important Note: For d > 0, subsequent culturing must have occurred in the same culture medium as prescribed above. (Calculators can be provided for other cell culture media and conditions. Please inquire.)
| d |
2. Enter N0, the total number of cells (live and dead) in the evaluation cultures at time = 0.
| N0 |
3. Enter N72, the total number cells (live and dead) after approximately 72 hours of culture.
| N72 |
4. Enter, in units of fractional days, T, the actual period of time between the N0 cell count and the N72 cell count.
| T |
Result:
Examples of Rapid-Counting Calculator Applications
Application for quantifying the HSC-specific fraction or dosage of CD34+-selected umbilical cord blood cells1. After isolation of a preparation of CD34+-selected umbilical cord blood cells, remove a sample of cells and perform a 72-hour evaluation culture as specified for HSC rapid-counting.
2. Enter the respective values of d (d = 0.0 days), N0, N72, and T.
3. Relate the HSC-specific fraction or dosage to other experimental factors or conditions under investigation.
Note: If the isolated cells are cultured with the conditions for rapid-counting evaluations, the same rapid-counting evaluation procedure will allow determination of the HSC-specific fraction at any time during the culture of the isolated cell preparation (i.e., for d > 0.0).
Application for Humanized Mouse Production
1. In parallel with SCID mouse injection of CD34+-selected umbilical cord blood cells, perform the HSC rapid-counting evaluation 72-hour cell culture. Or if you routinely perform a 72-hour expansion-culture before injecting cells, count the starting total cell number ("N0") and the 72- hour total cell number ("N72").
2. Evaluate the HSC rapid-count for correlation with engraftment efficiency. It is predicted to correlate with engraftment potency.
V. Asymmetrex® Rabbit Algorithm For Human Unfractionated Umbilical Cord Blood HSCs
This calculator allows determination of the tissue stem cell-specific fraction (SCF) of human HSCs in unfractionated umbilical cord blood when cultured in the following commercial cell culture medium after lysis or removal of red blood cells.Stemcell Technologies, StemSpanTM SFEM II (Cat#09655); StemSpanTM CD34+ Expansion Supplement (Cat#02691); pen/strep.
Culture and counting procedures
1. In a 24-well plate, initiate evaluation cultures with ideally 50,000 to 100,000 total viable cells per 2.25 mLs of the culture medium specified above. Fewer cells can be used, but more cells should not be used. Recommend preparing 6 replicate wells.
2. Use 3 of the cultures to determine the mean total cell count 4-5 hours after plating. This analysis sets time = 0 hours.
3. Approximately 72 hours later, determine the mean total cell count in the remaining 3 cultures, time = "72 hours." This period should not be less than 66 hours and not more than 78 hours (i.e., the culture period must be limited to the range in fractional days from 2.75 to 3.25).
Note: Accurate tissue stem cell counting requires an increase in total cell number during the period of this analysis.
Calculator Procedure
1. Enter, d, the number of days of serial culture from the first cryopreservation of the initial isolated primary tissue cell preparation (e.g., 0, 3.0, 6.0). Entry of fractional days is recommended for increased accuracy.
Important Note: For d > 0, subsequent culturing must have occurred in the same culture medium as prescribed above. (Calculators can be provided for other cell culture media and conditions. Please inquire.)
| d |
2. Enter N0, the total number of cells (live and dead) in the evaluation cultures at time = 0.
| N0 |
3. Enter N72, the total number cells (live and dead) after approximately 72 hours of culture.
| N72 |
4. Enter, in units of fractional days, T, the actual period of time between the N0 cell count and the N72 cell count.
| T |
Result:
Example of a Rapid-Counting Calculator Application
1. In parallel with plating of primary cord blood unit mononuclear cells for CFU assay, perform the HSC rapid-counting evaluation 72-hour cell culture.
2. Relate the HSC rapid-count to your CFU assay results.
3. Evaluate the HSC rapid-count for correlation with clinical outcomes. It is predicted to be a better potency measure than CFU.
VI. Asymmetrex® Rabbit Algorithm For Human Adipose-Derived Mesenchymal Stem Cells
This calculator allows determination of the tissue stem cell-specific fraction (SCF) of primary human adipose-derived mesenchymal stem cells when cultured in the following commercial cell culture medium:Thermo Fisher MesenPro RSTM Basal Medium supplemented with MesenPro RSTM Growth supplement (Kit Cat#1276012), 2 mM L-glutatmine, and pen/strep.
Culture and counting procedures
1. In a 6-well plate, initiate evaluation cultures with ideally 50,000 to 100,000 total viable cells per 5.0 mLs of the culture medium specified above. Fewer cells can be used, but more cells should not be used. Recommend preparing 6 replicate wells.
2. Use 3 of the cultures to determine the mean total cell count 4-5 hours after plating. This analysis sets time = 0 hours.
3. Approximately 72 hours later, determine the mean total cell count in the remaining 3 cultures, time = "72 hours." This period should not be less than 66 hours and not more than 78 hours (i.e., range in fractional days from 2.75 to 3.25).
Note: Accurate tissue stem cell counting requires an increase in total cell number during the period of this analysis.
Calculator Procedure
1. Enter, d, the number of days of serial culture from the first cryopreservation of the initial isolated primary tissue cell preparation (e.g., 0, 3.0, 6.0). Entry of fractional days is recommended for increased accuracy.
Important Note: For d > 0, subsequent culturing must have occurred in the same culture medium as prescribed above. (Calculators can be provided for other cell culture media and conditions. Please inquire.)
| d |
2. Enter N0, the total number of cells (live and dead) in the evaluation cultures at time = 0.
| N0 |
3. Enter N72, the total number cells (live and dead) after approximately 72 hours of culture.
| N72 |
4. Enter, in units of fractional days, T, the actual period of time between the N0 cell count and the N72 cell count.
| T |
Result:
Example of a Rapid-Counting Calculator Application
1. After isolation of experimental primary MSC-containing preparations, remove a sample of cells and perform a 72-hour culture as specified for MSC rapid-counting.
2. Enter the respective values of d (d = 0.0 days), N0, N72, and T.
3. Relate the MSC-specific fraction or dosage to other experimental factors or conditions under investigation.
Note: If the isolated cells are cultured with the conditions for rapid-counting evaluations, the same rapid-counting evaluation procedure will allow determination of the MSC-specific fraction at any time during the culture of the isolated cell preparation (i.e., for d > 0.0).
VII. Asymmetrex® Rabbit Algorithm For Human Mobilized Peripheral Blood CD34+ HSCs
This calculator allows determination of the tissue stem cell-specific fraction (SCF) of HSCs in human CD34+-selected mobilized peripheral blood (MPB) cells when cultured in the following commercial cell culture medium after lysis or removal of red blood cells.Stemcell Technologies, StemSpanTM SFEM II (Cat#09655); StemSpanTM CD34+ Expansion Supplement (Cat#02691); pen/strep.
Culture and counting procedures
1. In a 24-well plate, initiate evaluation cultures with ideally 50,000 to 100,000 total viable cells per 2.25 mLs of the culture medium specified above. Fewer cells can be used, but more cells should not be used. Recommend preparing 6 replicate wells.
2. Use 3 of the cultures to determine the mean total cell count 4-5 hours after plating. This analysis sets time = 0 hours.
3. Approximately 72 hours later, determine the mean total cell count in the remaining 3 cultures, time = "72 hours." This period should not be less than 66 hours and not more than 78 hours (i.e., the culture period must be limited to the range in fractional days from 2.75 to 3.25).
Note: Accurate tissue stem cell counting requires an increase in total cell number during the period of this analysis.
Calculator Procedure
1.Enter, d, the number of days of serial culture from the first cryopreservation of the initial isolated primary tissue cell preparation (e.g., 0, 3.0, 6.0). Entry of fractional days is recommended for increased accuracy.
Important Note: For d > 0, subsequent culturing must have occurred in the same culture medium as prescribed above. (Calculators can be provided for other cell culture media and conditions. Please inquire.)
| d |
2. Enter N0, the total number of cells (live and dead) in the evaluation cultures at time = 0.
| N0 |
3. Enter N72, the total number cells (live and dead) after approximately 72 hours of culture.
| N72 |
4. Enter, in units of fractional days, T, the actual period of time between the N0 cell count and the N72 cell count.
| T |
Result:
Examples of a Rapid-Counting Calculator Application
Application for quantifying the HSC-specific fraction or dosage of human CD34+-selected MPB cells1. After isolation of a preparations of human CD34+-selected MPB cells, remove a sample of cells and perform a 72-hour evaluation culture as specified for HSC rapid-counting.
2. Enter the respective values of d (d = 0.0 days), N0, N72, and T.
3. Relate the HSC-specific fraction or dosage to other experimental factors or conditions under investigation.
Note: If the isolated cells are cultured with the conditions for rapid-counting evaluations, the same rapid-counting evaluation procedure will allow determination of the HSC-specific fraction at any time during the culture of the isolated cell preparation (i.e., for d > 0.0).
Application for SCID Mouse Repopulating Cell Assay Analyses
1. In parallel with SCID mouse injection of human CD34+-selected MPB cells, perform the HSC rapid-counting evaluation 72-hour cell culture. Or if you routinely perform a 72-hour expansion-culture before injecting cells, count the starting total cell number ("N0") and the 72-hour total cell number ("N72").
2. Evaluate the HSC rapid-count for correlation with engraftment efficiency. It is predicted to correlate with engraftment potency.
VIII. Asymmetrex® Rabbit Algorithm For Human Adult Bone Marrow CD34+ HSCs
This calculator allows determination of the tissue stem cell-specific fraction (SCF) of HSCs in adult human CD34+-selected bone marrow (BM) cells when cultured in the following commercial cell culture medium after lysis or removal of red blood cells.Stemcell Technologies, StemSpanTM SFEM II (Cat#09655); StemSpanTM CD34+ Expansion Supplement (Cat#02691); pen/strep.
Culture and counting procedures
1. In a 24-well plate, initiate evaluation cultures with ideally 50,000 to 100,000 total viable cells per 2.25 mLs of the culture medium specified above. Fewer cells can be used, but more cells should not be used. Recommend preparing 6 replicate wells.
2. Use 3 of the cultures to determine the mean total cell count 4-5 hours after plating. This analysis sets time = 0 hours.
3. Approximately 72 hours later, determine the mean total cell count in the remaining 3 cultures, time = "72 hours." This period should not be less than 66 hours and not more than 78 hours (i.e., the culture period must be limited to the range in fractional days from 2.75 to 3.25).
Note: Accurate tissue stem cell counting requires an increase in total cell number during the period of this analysis.
Calculator Procedure
1.Enter, d, the number of days of serial culture from the first cryopreservation of the initial isolated primary tissue cell preparation (e.g., 0, 3.0, 6.0). Entry of fractional days is recommended for increased accuracy.
Important Note: For d > 0, subsequent culturing must have occurred in the same culture medium as prescribed above. (Calculators can be provided for other cell culture media and conditions. Please inquire.)
| d |
2. Enter N0, the total number of cells (live and dead) in the evaluation cultures at time = 0.
| N0 |
3. Enter N72, the total number cells (live and dead) after approximately 72 hours of culture.
| N72 |
4. Enter, in units of fractional days, T, the actual period of time between the N0 cell count and the N72 cell count.
| T |
Result:
Examples of a Rapid-Counting Calculator Application
Application for quantifying the HSC-specific fraction or dosage of adult human CD34+-selected BM cells1. After isolation of a preparations of adult human CD34+-selected BM cells, remove a sample of cells and perform a 72-hour evaluation culture as specified for HSC rapid-counting.
2. Enter the respective values of d (d = 0.0 days), N0, N72, and T.
3. Relate the HSC-specific fraction or dosage to other experimental factors or conditions under investigation.
Note: If the isolated cells are cultured with the conditions for rapid-counting evaluations, the same rapid-counting evaluation procedure will allow determination of the HSC-specific fraction at any time during the culture of the isolated cell preparation (i.e., for d > 0.0).
Application for SCID Mouse Repopulating Cell Assay Analyses
1. In parallel with SCID mouse injection of adult human CD34+-selected BM cells, perform the HSC rapid-counting evaluation 72-hour cell culture. Or if you routinely perform a 72-hour expansion-culture before injecting cells, count the starting total cell number ("N0") and the 72-hour total cell number ("N72").
2. Evaluate the HSC rapid-count for correlation with engraftment efficiency. It is predicted to correlate with engraftment potency.