Cell lysis

Macrophages were washed with PBS warmed to 37°C with a vacuum aspirator (Integra Vacusafe model 158-3xx). M. tuberculosis was sterilized on the cell culture plates by filling up each well of the OmniTray with 100% methanol at 4°C. The lids of the OmniTrays were also treated with methanol in order to fully sterilize all of the plate surfaces. Following removal of the OmniTrays in methanol from the biosafety level 3 facility, the cells were washed twice with PBS at room temperature and lysed with 6 ml of lysis buffer (8 M urea, 150 mM NaCl,100 mM ammonium bicarbonate, pH 8; added per 10 ml of buffer: 1 tablet of Roche mini-complete protease inhibitor EDTA free and 1 tablet of Roche PhosSTOP tablet) at room temperaeture prepared fresh before each replicate. Cell lysis was performed by adding 4 ml of the lysis buffer to each of the four wells of an OmniTray, scraping the cells and lysis buffer to the bottom of the wells with a cell scraper, and then transfering the lysates to the next OmniTray until all of the wells from a given condition were lysed. The remaning 2 ml of lysis buffer was used to harvest residual material left on the plate surfaces using the cell scraper as previously described. Lysates were stored at -80°C until further processing.

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