Welcome to Asymmetrex’s kinetic stem cell (KSC) counting page! In addition to free KSC counting, this page begins with calculators for three useful parameters for tissue cell culture research: PDT, population doubling time; cell doublings; and CPD, cumulative population doublings. These factors are also the inputs for Asymmetrex’s KSC counting platforms. Use CPD data to get a TORTOISE TestTM calculation of tissue stem cell-specific number. Afterwards, obtain rapid stem cell counts using only simple PDT data with RABBIT CountTM algorithms.
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
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
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:
IV. Counting Tissue Stem Cells with Asymmetrex’s TORTOISE TestTM KSC Counting
A. Cells, culture medium, and equipment
1) Primary vertebrate tissue cells (pre-senescent, non-immortalized)
2) Culture medium in which the cells will proliferate (with any additives you want)
3) Any culture format you like – adherent, suspension, microcarrier
4) A cell culture incubator or bioreactor with any culture conditions you want
5) A way to count live cells and dead cells (electronic or manual)
It is important to get accurate and reliable cell counts. So, do triplicate sampling to determine the mean and standard deviation of each culture count.
B. Start your cell cultures
Put some cells into two culture vessels to start two replicate cell cultures. How many cells? Whatever you want, but generally 50,000-100,000 total live cells per 25 cm2 area for adherent cultures or per 2 mL of suspension culture. A single serial cell culture is sufficient for a KSC count analysis, but greater statistical confidence is achieved with three replicate serial cultures and a respective triplicate culture determination of the initial cell count (i.e., starting with 3 x 2 = 6 replicate cultures; See C below.).
C. Get an initial viable cell count
Two to 5 hours after starting your two cultures, take one of them, harvest the cells, and determine the total live cell and total dead cell count for the culture. This your Initial Cell Count. (If you are conducting a triplicate analysis, count three initial cell cultures in this manner.)
D. Serial culture and counting until reaching a terminal division arrest
During culture incubation, avoid moving or disturbing cultures. Every three days do the following:
1) Record the date and time with quarter hour precision.
2) Inspect your cultures by phase contrast microscopy. Record any anomalies (e.g., detachments)
3) Note the cultures’ cell health and, if adherent, degree of confluency, which will decrease with passaging.
4) Harvest the cells, resuspend them well, and transfer 1/20 of the total harvested cells to a new culture vessel of the same type with the same total volume of the same type of culture medium.
5) Use the non-transferred cells to determine and record the total live cell and total dead cell number for the passaged culture. Do not delay the counting in ways that would artificially increase dead cells.
E. Define terminal division arrest by one of two possible events
Natural terminal division arrest – The total number of cells at the end of a passage is not greater than the number of cells that were transferred to start that passage. When two consecutive passages show a natural arrest, serial culture can be stopped. Dilution terminal division arrest – By your most sensitive counting procedure, no cells are detected at the end of a passage. When two consecutive passages show a dilution arrest, serial culture can be stopped. The most sensitive counting procedure would be pelleting all of the non-transferred cells by centrifugation and resuspending them in sufficient volume for a minimum of three replicate sample counts.
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