I need to study some beneficial effect of different potential probiotics strains on HEK cells. I would like to know the best way to prepare these probiotics samples to test them on mammalian cell cultures. I have been checking on the literature and the most usual protocol is collect the supernatant of the culture media by centrifugation and neutralized it to pH 6-7.0 by the addition of NaOH and filter it by 0.22 um. So, with this protocol, the probiotic supernatant would be dissolved in the corresponding bacteria broth media but I would prefer dissolve it in the corresponding cell assay buffer. I saw some protocols that include lyophilization to concentrate it. I think that after lyophilization I may be able to dissolve it in the cell assay buffer. Any better suggestion?
Also, for the preparation of the microbial cell extract the residual bacterial cells pellet is washed three times with PBS (1x) and suspended in 3 ml of PBS. The suspension is subjected to a series of freeze-thaws (3x) and brought to 5 ml with PBS for sonication. The sonicates are centrifuged (at 14, 000 rpm for 10 min) and filter-sterilized before used in the following assays. Any other protocol?
Then I am wondering if should I also try alive bacteria cells on the cell culture screening? would the bacteria survive in usual cell culture buffer assays such as HBSS?
Hi Ami,
Thanks a millions for your reply! :) So, would you suggest me lyophilization of the supernatant or not? I saw most of the protocol dont include this step but it may be the best to get higher concentration of the possible bioactives released from the bacteria and also to re-dissolve it in assay buffer instead bacteria broth media?
Many thanks again for replying me,
Cristina
Many thanks again Ami! I will try both ways, so I can compare also.
Kind Regards,
Cristina
Hi Cristina ,
I am facing the same problem .Please let me know which method worked for you.What was the concentration you used in case of lyophilzed supernatants.
regards,
Manpreet
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