I am doing research on autophagy with bacteria. According to our lab protocol, first we leave the cells (RAW264.7) infected with bacteria for an hour at MOI=10, then wash out the cells in order to take away the bacteria in the supernate. After that we put the cells back into incubators to be continually infected for 1 h, 6 h,1 day, 2 days, and 3 days. In my results, the 1 hr cells only have less than 50 CFU.
It is very low in our lab, and I am wondering whether I have done something wrong. Is it because of my cell condition? RAW is supposed to be a macrophagy cell line and have a strong engulf ability. Why are my cells weak in swallowing?
Can someone answer my question and give my some help? Thank you!
What bacteria are you using? in my experience M.smegmatis infected RAW 264.7 cell lines are quite efficient in uptake of bacteria. I used similar strategy of infecting with bacteria for 1 hr and then washing of excess bacteria and then allowing the infection to progress. Is it possible that cell are dying immediately after infection and washed of with the bacteria within an hour?It is highly unlikely....do check it though.
Miao Lee, Try centrifuging the plate if you are not doing so. You can centrifuge at 300 g for 2 minutes. Bacteria can easily swim away from the cells.
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