I want to prepare conditioned medium from Jurkat cells.
I will culture Jurkat cells in normal culture media, then activate it with PMA 50ng/ml+Iono 1ug/ml
what should I do next？
Is there a protocol?
To prepare conditioned medium from Jurkat cells, you can follow the protocol below:
1. Culture Jurkat cells in an appropriate sized flask (such as T-75 cm^2 or T-175 cm^2 flasks) under normal culture media (RPMI 1640 + 10% FBS + 1% penicillin/streptomycin) until they reach 70-80% confluency.
2. Remove the culture media and wash the cells with PBS.
3. Add fresh culture media containing PMA (50 ng/ml) and Ionomycin (1 μg/ml) to the cells.
4. Incubate the cells for 24-48 hours at 37°C in a humidified incubator with 5% CO2.
5. Collect the conditioned medium by centrifugation at 3000 rpm for 10 minutes to remove cell debris.
6. Filter the supernatant through a 0.22 μm filter to remove any remaining cell debris.
7. Aliquot the conditioned medium and store it at -80°C until use.
There are also several kits available for preparing conditioned medium from cells, such as the Gibco Cell Culture Supernatant Processing Kit and the Thermo Scientific Pierce Concentrators. These kits can simplify the process and provide higher yields of conditioned medium.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus