I am trying to use CBMCs to for HHV6A infection.
What is the culture protocol?
What serum to use and media and at what concentrations?
Can I use freshly acquired CBMCs for this work?
Human herpesvirus 6 (HHV-6) like other herpesviruses, expresses sequentially immediate early (IE), early, and late genes during lytic infection. U94 play a role in the maintenance of the latent state, lymphoid cell lines stably express U94. HHV-6 is able to infect these cells, but viral replication is restricted with no cytopathic effects. JJhan cells are grown in suspension at 37°C in RPMI 1640 medium supplemented with 10% fetal calf serum. Cell-free viral inocula can be obtained by pelleting 250 ml of cell cultures infected with HHV-6A (strain U1102) exhibiting complete cytopathic effect (CPE). Infected cells, resuspend in 2 ml of fetal calf serum supplemented with RNase (Boehringer Mannheim, 50 μg/ml), get disrupted by four cycles of freezing in liquid nitrogen and thawing at 37°C. The resulting inoculum is completely free of living cells, as checked by microscopic observation and cultivation. It is analyzed by reverse transcription–PCR (RT-PCR) (both for β-actin and for a panel of viral mRNAs) to ensure that RNA is completely absent. Infection then is performed by resuspending 107 cells in the resulting viral inoculum. After 1 hr of adsorption at 37°C the cells are diluted with fresh medium to attain a final concentration of 5 × 105 cells/ml
Thank You Dr. Upadhyay. I am currently using the same technique you have mentioned. But 250 ml of infected cell culture is a huge amount to start with to obtain cell free virus. I was wondering if we could get cell free virus using CBMCs as quite a few other labs in the world are doing it to get cell free virus.
If you want to use CBMCs for the infection, it'sbetter to stimulate the CBMCs with IL-2 and PHA for 3-4 days. Using 1 ml high titre virus you can effectively infect 5x10^6 cells in 7 ml 10 % FBS RPMI medium.
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