lysis of cells - how to lyse cells (Jun/05/2006 )
hi,
can somebody please tell me exactly how to lyse cell cultures during adhesion/invasion assay in detail? like how much detergent is added, and then how much media is added? how is mixed and so on.....
thank you for the time.
what specifically are you trying to do?
we look at invasion and adherence of S aureus into keratinocyte cultures. We infect with the bacteria and then at given timepoints, the monolayers are trypsinized briefly, removed to tubes, and sterile water is added. they sit at room temp for a bit for hypotonic lysis to occur, then dilutions are plated to get the bacterial counts (for internalization/invasion assay, we kill the extracellular bacteria after a given time, then allow further incubation)
detergent is never added in this protocol, you may be looking for something else?
we look at invasion and adherence of S aureus into keratinocyte cultures. We infect with the bacteria and then at given timepoints, the monolayers are trypsinized briefly, removed to tubes, and sterile water is added. they sit at room temp for a bit for hypotonic lysis to occur, then dilutions are plated to get the bacterial counts (for internalization/invasion assay, we kill the extracellular bacteria after a given time, then allow further incubation)
detergent is never added in this protocol, you may be looking for something else?
thank you for the reply. Specifically, I am trying to lyse INT-497 cells after invasion assay to count intracellular bacteria. Literature indicates that cell are mostly lysed in 0.1-1% triton x-100 (a detergent???). I was just wondering the details of this last step. like what the solvet is for 1% triton (water, pbs ect.), how much triton is added to the cells. for how long? and whatever the details are afterwards.
thanks.
I'm sorry, I am unable to help you. we use water to lyse our cells to ensure that the bacteria are not affected.
perhaps someone else knows more about your cell type?
umm not that i can be all that much help, but triton X is a mild detergent, good for lysing cells, usually i make up a 1% triton-x solution in PBS, although it might vary for your needs
[quote name='aimikins' date='Jun 6 2006, 04:59 PM' post='54442']
I'm sorry, I am unable to help you. we use water to lyse our cells to ensure that the bacteria are not affected.
perhaps someone else knows more about your cell type?
thank you anyway.
This is the method we have used for Salmonella infection in J774 Cells.
J774 in 6 well plates (- Antibiotics), and leave overnight.
Grow Salmonella for 3 hours (log phase), spec and then infect J774 at MOI of 0.1, 1, 10, 100.
Leave for time course of experiment, at 0, 1, 2, 4, 8, 16, 24hrs.
At time point, take off cell supernatant and wash monolayer x1 with PBS and add to discarded cell media and spin at 300g for 5 minutes. Take off supernatant and lyse cells with distilled water (100ul). This will give you the number of bacteria in the extracellular space.
Wash cell monolayer x 2 with PBS and then add distilled water to lyse cells. Spin as above, this will give you the intracellular bacteria number.
J774 are macrophages so they actively phagocytose the salmonella.