Live dead baclight bacterial viability kit

Integrity of E. coli BW25113 cells inhibited by other E. coli BW25113 inside a square steak was determined using Live/Dead BacLight Bacterial Viability Kit (L13152, Sigma) with some modification. After a 24-h incubation, E. coli BW25113 cells (2 μL of 5*10⁷ CFU/mL) were spotted in the center of the square streak and incubated at 37 °C for 90 min or onto fresh LB 0.8% agar as a control. The spotted E. coli BW25113 cells were stained by adding 10 μL of 2 × concentration of SYTO9 (12 μM) and Propidium Iodide (60 μM) on the spotted bacterial cells and incubated at 21 °C in the dark for 15 min. This process was repeated one more time to ensure sufficient staining. The agar with stained inoculant was cut from the plate and placed inverted in a glass bottom petri dish (P50G-1.5–30-F, Matek) so that spotted bacterial cells are facing the glass bottom. Bacterial cells were imaged on a Leica SP5 laser confocal microscope using a 63 × water immersion objective (HCX PLO APO CS 1.20 NA), 3 × digital zoom, and sequential scanning at 488 nm/500–530 nm (excitation/emission) for SYTO9 and 543 nm/604–700 nm Propidium Iodide.

aIIbb3 inhibition: Abciximab (C7E3-Fab) from Janssen and Tirofiban from Ibigen, myosin IIa inhibition: blebbistatin(−) (Cayman Chemical) (50 µM); blebbistatin(+) (Cayman Chemical) was used as a negative control. To avoid fluorescence-induced toxicity, para-nitroblebbistatin (50 µM, Optopharma) was used in a subset of experiments. Arp2/3 complex inhibition: CK666 (200 µM) and negative control: CK689 (both Merck Millipore). Actin polymerization inhibition: cytochalasin D (2.5 µM) (Sigma). Other: carboxyfluorescein succinimidyl ester (CFSE), Annexin V, busulfan, plasmin, thrombin, Fluo-4 AM, LPS O111:B4, CCL-2 (Sigma) iron (III) chloride (FeCl₃), casein, recombinant human albumin (HSA), fibrinogen from human plasma, unconjugated and conjugated to AlexaFluor 488 and 594, Rhodamine 6G, adenosine diphosphate (ADP), NP40, polyethylene glycol 400 (PEG400), phalloidin-AlexaFluor 488, hexamethyldisilazane (HMDS), paraformaldehyde (PFA), GDA, and LIVE/DEAD BacLight Bacterial Viability Kit were purchased from Sigma-Aldrich. Horm collagen was from Takeda, prostaglandin I2 sodium salt from Abcam, and U46619 from Tocris.