Good day, I will be receiving a stock of THP-1 passage number 6 and would need to prepare the culture media. The culture media will contain the base medium RPMI 1640, 10% FBS and 1% penicillin-streptomycin. I need to expand these cells to have a future bank of cells.
However, do I need to heat-inactivate the FBS if I want to expand the cells?
If so, doesn't heat-inactivation degrade growth factors needed for the cells to grow?
In the future, I would be infecting these cells with a stock of virus, so is heat-inactivation required?
Thank you
Cordially, Eduardo Alvarez.
You should heat inactivate your FBS otherwise the active complement protein might kill your cells or affect the growth pattern. If your commercially purchased FBS is not heat inactivated then please follow the steps for heat inactivation.
- Heat your FBS at 56deg celsius for 30minutes before using for cell culture.
Hope this will work.
Hello Eduardo.
Yes, it is absolutely essential. In addition to Gundappa's comment, just centrifuge the inactivated serum at 2,500 rpm for 10 min. Take the supernatant fraction for storage or immediate use. This will remove aggregated proteins and particles that will ease subsequent milliporing.
Regards.
I never have cultured that particular cell type, but in general the need for heat inactivation is overblown and often unnecessary.
If you do decide to heat inactivate, then you cannot simply put the serum in a 56C water bath for 30 minutes. That will accomplish nothing, as the actual temperature of the FBS is unlikely to reach 56C within those 30 minutes.
You have to use a temperature probe in a water container that is the same size and material as what is holding the FBS. The inactivation timing only commences when the contents reach 56C.
The containers must be sufficiently submerged to prevent a significant temperature gradient in the containers. The probe and FBS containers must start out at identical temperatures. Your water bath has to be able to maintain sufficiently stable temperatures such that the containers can reach and stay above 56.0C but not go over 56.5C. For that reason you should do at least one practice run using an equal volume of water in containers to match the volume of FBS that you plan on inactivating. This will tell you approximately how long the entire process will take and will let you know if your water-bath is viable for the task. Many are not.
Thank you all for your help. I will heat-inactivate the FBS. Take care.
If you choose to inactivate your FBS, I suggest that you do comparative cultures of both types of FBS.
Hi,
I cultivated THP-1 in RPMI + FBS HI however ATCC reports the following:
To prepare a bottle of complete growth medium, add the following:
- 500 mL RPMI-1640 (ATCC 30-2001)
- 56 mL FBS (ATCC 30-2020) – not heat-inactivated
Note: Because of limited stability, the following components should be added to an aliquot of the above culture medium fresh prior to seeding or performing fluid additions. Do not store media supplemented with 2-Mercaptoethanol
Gibco 2-Mercaptoethanol (ThermoFisher Scientific Catalog # 21985-023): use 0.9 µL/1 mL culture medium.
The FBS should not be heat-inactivated. If growth is slow try using a different FBS and/or increasing the FBS concentration temporarily to 20%.
Hope it helps,
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