I took new ordered sf9 cells from the vendor and I did not following steps: 1, take the cells out from the vial, thaw, and add to a small stationary dish flask. The first day I can see majority of cells floated on the surface of the medium. A few cells attached to the bottom of the dish flask. 2, after 3-4 days, I removed the medium with the dead cells, and add fresh new medium. 3, after 3-4 days, the live cells grow until about 80% confluence. 4, I took a scraper and scraped the cells into medium. 5, I transfered the cells in the medium to the shaker flask. 6, I checked the number of cells in the shaker flask but the concentration is low, less than 1*10^6 cells/ml, even after 2-3 days of growth. So the problem is, the cells grow slower than expected. Can you please let me know if there is anything wrong with the previous steps? Thanks. hai
Since usual hormone assays based upon affinity of immunoglobulins (ELISA) are not quantitative at all, the study by using hormone should be performed by using reliable HPLC method. CLIA (Chemiluminescent Immunoassay) method and other immunoassays are also sadly making many erroneous results. About 2,000-fold difference has been occurred between HPLC method and notorious ELISA method (please see files; JCB fucoidan transport, Tokita ELISA, and ELISA fucoidan). Then, estradiol-17β concentration in female serum measured by immunological method (160 pg/mL; 0.59 nM) may become 0.32 μg/mL (1.17 μM). The reliable HPLC method for hormone assay has also been mentioned recently (please see file; The Fascio Effect). As an example, serum free-biotin concentration, which has not yet been considered as hormone but has a similar growth factor (vitamin H) as hormone, has been determined as 0.122 μg/mL (0.499 μM) by using reliable HPLC method (please see file; Wide range of Biotin).
Then, please use the biotin-added culture-medium as shown in the file "JCB Fucoidan transport". 5 μg/mL (20 μM) of free biotin has been added to the medium, since human serum has 15-fold higher amount of total-biotin (bound-form biotin) than free-biotin. Since biotin (vitamin H) is a very effective growth factor also to fungus, please use Amphotericin B in order to prevent the infection or contamination of fungus.
To your steps:
1, do you get rid of the freezing agents? Centrifuge the cells and take them up in new media (add sufficient FCS)
2, I would remove dead cell and supply with fresh media already after one day.
3, all good
4, I usually just rinse the cells off the surface (with the pipet) and do not use a scraper. Then I supply the remaining cells on the surface with fresh media to have a back-up culture. Usually, I first transfer them to another static culture until I reached a sufficient growth in T-flasks.
5, what material is the flask? Glass or plastic? How strong are you shaking?
6, what is the viability of the cells?
- I would not worry if the cells are growing a little slower for the first 1-2 weeks until you have a stable culture with a good viability (>96%).
- Make sure that you often change the media to get rid of the freezing agents, most of them are toxic or growth inhibiting.
- Add at the beginning more FCS then usually, the cells like it.
to maintain cell growth is usually done special treatment, such as aeration with shaker and may also require additional nutrients, as well as growth temperatures can give effect in the growth of bacterial cells.
the cell development phase, ie 3-4 days is the adaptation of the growth environment, then into the development phase or enlargement of cell size, days 5-12 is the period of cell division, and then the stationary phase, meaning the number of dead cells may be relatively equal to the number dividing cells. and further into the cell death phase usually in days 20 and above. But this condition is very relative by every cell, hence nutrition and other treatment need to be paid attention
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