Before use in flow cytometry, MФs were infected as previously described, washed five times with PBS for 5 min each, and collected into 1× Extraction Buffer with protease inhibitor cocktail (Sigma Aldrich).
For intracellular staining, cells were first stained with cell surface mAb as described, washed, and then fixed with 4% paraformaldehyde for 15 min and permeabilized in PBS, 2% FCS, 0.5% saponin and incubated with Perforin-FITC (BD Pharmingen, Heidelberg, Germany) and Granzyme B PerCP (Caltag laboratories) for 30 min at 4°C.
Lister described washing wounds with the acid to destroy "septic germs".
Cells were stressed as described above, washed with PBS and stored at −70°C until use.
MФs were infected as described above, washed with 4°C PBS, and incubated in a solution of organelle-specific 1× Extraction Buffer (Organelle Isolation Kits®, Sigma Aldrich) and a 0.1% solution of protease inhibitor cocktail (Sigma Aldrich) overnight (ON) on ice at 4°C.
Cells, transfected or treated as described, were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature.
For internalization studies, cells were grown in 12-well plates, treated with siRNAs as described above, washed with MEM supplemented with 20 mM, HEPES pH 7.4 and 0.1% BSA, and incubated in a water bath.
Wandering stage third instar larvae were collected as described above washed with fresh water and then they were bled into 20 µl PBS by ripping the cuticle with forceps.
After the period of infection macrophages were harvested as described above, washed with PBS and fixed and permeabilized using a "Fix & Perm kit" (Pharmigen); 1 × 105 cells were centrifuged on to glass slides with a Cytospin.
CasKi and 2A3 cells were grown as described above, washed with sterile PBS, placed into Eppendorf tubes in the amount of 10 cells per tube in 1 ml PBS and incubated for 3 h at 37 °C with 20 μCi Re-C1P5 mAb or left untreated.
