Hi everyone!
I want to perform a killing assay using peritoneal macrophages from peritonitis caused by a injection of S. aureus.
Does anyone know how to perform it, using a plate 96-well and MTT?
I read protocols, but I am really confused and lost about the assay.
If anyone could suggest something to me I really appreciate that. Thank you.
i think most often used study is counting CFU on a plate; for invasion and killing assays.
In 96 well format its very difficult assay..but you might try this also.
I call it as rescue assay
Since you mentioned, you give injection to mice with specific MOI dose; then after you isolate peritoneal macrophages , seed in 96 well plate.(Which means infection invivo)
Now isolate the cells , put them in 96 well plate incubate in media containing Gentamycin (its an antibiotic kills only extracellular bacteria, cant ablr to permit inside of a cell) for 30 min.
Now you can lyse the cell with only 20 ul of 0.001% SDS for 30 sec add 80 ul of Culture medium with out any antibiotic.
Now add 10 ul of alamar blue to it, wait for 3 hrs to grow them back,, if really bacteria killed they wont grow ,,if not they might grow.
You can read either OD or fluorescence with alamr blue
gud luck
Hi Srikanth Aluru, how u doing?
I searched but i did not find any paper describing the methodology. Do you have one to suggest?
Thank you!!
i am doing good.
I have seen above protocols in Jove
title:
A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line
I am sure the concentration of this SDS doesnot kill any intracellular bacteria , but effectively lyse the cell.
also check ,,extracellular Gentamicin assay,,
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