I am currently working with Chlamydia infected HeLa cells. The protocol given to me by my PI states this should be happening within 2-10 min. However, he and I have given it a trial run and even after 45 min, we did not observe any lysis happening (under microscope). The basic run down of the protocol is to remove media, wash cells in with DI water, discard, then add ~2 mL of water per well (6 well plate) and observe under microscope. I am currently going through a couple more trials and am thinking of repeating the washing step and adding some form of mechanical shearing.
Is our estimated time too optimistic? Is there something I'm missing?
Thank you.
It is very intriguing that your cells did not release its contents in distilled water, which is hypotonic to cells. One possible explanation is that your cells are not washed appropriately, thus, water is not hypotonic. I would wash cells with large excess of water and then incubate in highly distilled water. It should not take more than 30 min for cell lysis to occur. If you still do not see cell lysis, other possibilities such as the quality of water may need to be considered.
Thank you Dr. Choubey.
The washing turned out to be the main problem. After 2 rounds of washes I was able to observe initial lysis at about 25 minutes (up to ~45min for 90% lysis). I assume another round of washing would further shorten the time but I decide to stick with 2 washes to limit cell loss and have some buffer time to prepare for the next portion of the protocol.
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