Axioobserver a1 inverted fluorescence microscope

After RAPA treatment (0, 2, or 5 nM) for 0, 7, 14, and 21 d, BMSCs were fixed in 4% paraformaldehyde, permeated with 0.2% Triton X-100 (pH 7.4) for 10 min, and blocked with 5% bovine serum albumin. The cells were subsequently incubated with an anti-LC3 antibody (Proteintech, China) overnight at 4 °C and treated with a fluorescent secondary antibody (Abcam, USA) for 1.5 h. Next, the nucleus was stained using DAPI ( Beyotime, China). Finally, images were acquired with an Axio Observer A1 inverted fluorescence microscope (Carl Zeiss, Germany).

Cells (CHO-native (45,000) or DLD-1 (30,000) cells/well) were seeded out in an Ibidi 8-well chamber slide (Ibidi, Gräfelfing, Germany) 1 day before the start of the experiment. CHO-native cells were washed twice with PBS and incubated with the platelet-derived microvesicle suspension derived from a total of 1.2 ml of whole blood or Stx2:Alexa555 200 ng/mL, corresponding to the concentration used to generate the Stx2-positive platelet microvesicles.
DLD-1 cells were incubated with the microvesicle suspension derived from 350,000 HeLa cells, or Stx2:Alexa 488 at 31 ng/mL, which gave the equivalent fluorescence as the microvesicle suspension when measured in the Glomax Discover System.
Microvesicles and labeled Stx2 were diluted in Opti-MEM (Invitrogen, Carlsbad, CA) and incubated with the cells for 4 h. After the incubation, cells were fixed in 4% paraformaldehyde (Histolabs, Västra Frölunda, Sweden), for 20 min. CHO-native cells were stained with NucBlue Live Cell Stain (Thermo Fisher Scientific) and DLD-1 cells were stained with Cellmask Deep Red plasma membrane stain (1.6 μg/mL, Thermo Fisher Scientific) and visualized in an Axio Observer.A1 inverted fluorescence microscope (Zeiss) or in a Ti-E inverted fluorescence microscope equipped with a Nikon structured illumination microscopy module (Nikon Instruments, Tokyo, Japan).