Diagnostic microscope slides

Fat body dissections were performed on flies expressing GFP-mCherry-Atg8a. Flies were cut transversally with a scalpel on a petri dish cleaned with 70% ethanol to observe the fat bodies. Fat bodies were fixed with PFA 8% and mounted on diagnostic microscope slides (Thermo Fisher Scientific) in Vectashield with DAPI (Vector Laboratories).
To observe hemocytes, wild-type larvae injected with untreated or heat killed spores (treatment at 100°C for 15 minutes) were opened 6 hours after injection in a drop of 1x PBS directly on diagnostic microscope slides. After dissection, samples were left for 30 minutes to settle the cells on the slides. Hemocytes were fixed with 8% PFA, permeabilized for 15 min with 1x PBS and 0.1% Triton X-100. Samples were blocked for 2h in 1x PBS, 0.1% Triton X-100 and 2% BSA (PTB). Hemocytes were incubated in PTB plus the primary rabbit antibody anti-Tr spores (1/500) and 10 μM of FITC phalloidin (Sigma-Aldrich #P5282). Samples were washed for 15 min with 1x PBS and 0.1% Triton X-100. Cells were incubated for 2h on PTB plus the secondary goat antibody anti-rabbit coupled to Cy3 (1/500) (Invitrogen #A10522). Hemocytes were washed for 15 min with 1x PBS and 0.1% Triton X-100 and mounted in Vectashield with DAPI (Vector Laboratories). All samples were observed using a LSM 780 confocal microscope (Zeiss).

1x10⁵ cells per sample were starved in 0% FBS RPMI and rested on diagnostic microscope slides (Thermo Fisher Scientific) for 1 h at 37°C. Cells were left unstimulated or stimulated with 5 μg/ml anti-CD3ε (OKT3) for 5 min at 37°C. Cells were then fixed in 2% PFA for 15 min, permeabilized with 0.5% saponin for 30 min and blocked. Blocked cells were stained according to the manufacturer’s instructions with the Duolink kit (Olink Bioscience) with goat anti-CD3ε (Everest Biotechnology) and mouse anti-LCK (3A5, Santa Cruz Biotechnology). Nuclei were stained with DAPI (Roth). A total of 5–7 images (on average 700 cells) per sample were taken at ×60 with a confocal microscope (Nikon C2) and analyzed with BlobFinder.

Conjugates between Jurkat cells and SEE-pulsed Raji B cells were carried out as previously described (Finetti et al., 2009 (link)). Raji cells were pulsed for 2 h with 10 μg/ml SEE (Toxin Technology, Sarasota, FL, United States) and labeled with 10 μM Cell Tracker Blue (Molecular Probes) for the last 20 min. Conjugates between T cells and unpulsed B cells were used as negative controls. SEE-pulsed or unpulsed Raji B cells were mixed with Jurkat T cells (1:1) and conjugates analyzed 15 min after their formation. Samples were allowed to adhere for 15 min on poly-L-lysine (Sigma-Aldrich)-coated wells of diagnostic microscope slides (ThermoFisher Scientific), then fixed by immersion in methanol for 10 min at -20°C. Following fixation, samples were washed in PBS and incubated with anti-pTyr (Cell Signaling, #8954) at 10 μg/mL and anti-CD3ζ at 15 μg/mL (SantaCruz, #sc-1239) in PBS 1X overnight at 4°C. After washing in PBS, samples were incubated for 45 min at room temperature with anti-rabbit Alexa-Fluor-488- and anti-mouse Alexa-Fluor-555-labeled secondary antibodies (ThermoFisher Scientific, #A11008 and #A211422, respectively).