Evos 2 fl

After transfection with indicated plasmids or bacterial infection, Cells were washed with 1×PBS twice, then fixed with 4% paraformaldehyde (PFA) or cold methanol for 20 min, after that they were permeabilized by 0.2% Triton X-100 for 15 min. Subsequently, cells were immunostained with anti-tubulin mouse antibody (1:200, Abcam, USA), anti-GEF-H1 rabbit antibody (1:150, Abcam, USA), anti-FLAG mouse antibody (1:200, Genscript, China) or anti-Paxillin rabbit antibody (1:200, Abcam, USA) overnight at 4°C. Secondary antibodies for immunolabeling include AlexaFluor 488-labelled anti-mouse IgG antibody (1:250, Yeasen, China), AlexaFluor 594-labelled anti-rabbit IgG antibody (1:250, Yeasen, China). For actin staining, cells were labeled with TRITC-phalloidin (1:200, Yeasen, China). Plasma membrane was probed by using 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC18(3), Beyotime, China). Golgi apparatus was probed by Golgi-Tracker Red (Beyotime, China). Mitochondria was visualized by Mito-Tracker Red CMXRos (Beyotime, China). Endoplasmic reticulum was detected by ER-Tracker Red (Beyotime, China). Images were captured using a confocal laser scanning microscope (Leica, Germany) or epi-fluorescence microscope (EVOS FL-2, Life technologies, USA).

For the apoptosis assay, YO-PRO-1 reagent can be used as a sensitive indicator of the apoptosis [46 (link)]. Continuously increasing green fluorescence alone indicates apoptotic cells, while the simultaneous red and green fluorescence indicates dead cells. Here, we use YO-PRO^TM-1/ Propidium Iodide (PI) (Vybrant Apoptosis Assay kit 4; Thermo Fisher, Waltham, MA, USA) to assess the apoptosis of S. aureus-induced MAC-T cells. In brief, 1 × 10⁵ MAC-T cells per well was seeded in 96-well culture plates, then washed three times and infected by 1 × 10⁶ cfu/mL of S. aureus (MOI = 10) or plus with scFvs (final scFv or mixture concentration was 40 μg/mL) at a range of time points. Next, 100 μL of standard staining working solution was added to the respective well in darkness at 37 °C for 20 min. After being dyed, the stained MAC-T cells were visualized under the epi-fluorescence microscope (EVOS FL-2, Life technologies, New York, NY, USA) [47 (link)].

Macrophages were stimulated with vehicle DMSO (Sigma Aldrich, D2650) or WY14643 for 60 minutes, then biological triplicates were infected at an MOI of 20 with live Staphylococcus aureus Newman-mCherry for 30, 60, 90, or 120 minutes. 24 well plates were immediately centrifuged at 200 x g for 5 minutes. Macrophages were stained with CellMask™ Orange (#C10045) just prior to infection, and Sytox™ Green (S7020, 0.4mM) to detect bacterial killing. Imaging was done on EVOS 2 FL (Invitrogen) and analyzed using HCS Cell Studio Analysis Software (ThermoFisher).