Technical Library
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The population doubling time or generation time (tg) is usually calculated during the logarithmic phase of growth. It specifies the time (t) in hours needed by the culture to double its cell number.
tg = t / n
n: number of population doublings
PromoCell cell pellets are made from > 1 million cells and are dissolved in 200 µl RNAlater.
The majority of PromoCell cell pellets (C-14**) are prepared 1 passage after thawing the cryopreserved cells.
Examples:
HUVEC and HUAEC correspond to P1 after thawing; therefore the pellets are frozen in P2.
SMC or keratinocytes or epithelial cells correspond to P2 after thawing; therefore the pellets are frozen in P3.
In contrast; our blood cells are cryopreserved directly after cell isolation. These pellets are prepared after thawing the cryovials with no further cultivation step.
The recommended media for a particular cell type are specified on the respective product page ("Recommended Products") and can be found in the Manual belonging to the cells.
The recommended Growth Media are specified in the Manual belonging to the cells and can be also found on the product page of the cells ("Recommender products").
Yes you can use accutase to detach Normal Human Cells. Accutase acts very gently on the cells. Cell membranes and surface epitopes will not be harmed. It is therefore mostly used for applications that require unchanged surface markers e.g. for flow cytometry or for detachment of very sensitive cells.
PromoCell recommend to trypsinize all Normal Human Cells at room temperature and to monitor the detachment under the microscope. Prolonged trypsinization at 37°C can lead to irreversible damage of the cells.
Yes but the experimental starvation conditions have to be determined individually for each cell type. Usually the cells are maintained in basal medium with reduced growth factor concentrations or lower FBS content. The cells need to be in a good condition and the starvation should be kept as short as possible as prolonged serum and/or growth factor deprivation induces apoptosis.
Most commercially available trypsin solutions have trypsin concentrations of 0.05% or higher which can harm the primary cells. PromoCell recommend using a 0.04% trypsin / 0.03 % EDTA solution for the subculture of Normal Human Cells.
The PromoCell trypsin 0.04% / EDTA 0.03% solution and the PromoCell TNS solution are both based on HepesBSS. Therefore it is best to also use HepesBSS to wash the cells prior to trypsinization.
We recommend to use the DetachKit when subculturing our Normal Human Cells. It contains a HepesBSS washing buffer; trypsin 0.04% / EDTA 0.03% solution; and TNS; a trypsin inhibitor from soybean.
Please note: Many of our culture media have low serum content or no serum at all. These media are not suitable to inactivate trypsin during subculture.
Yes RNAlater Solution will denature proteins. Therefore protein obtained from PromoCell cell pellets will be suitable for applications such as Western blotting or 2D gel electrophoresis but not for applications that require native protein.