Eclipse ti e confocal microscope

Cells were plated into Ibidi μ-slides VI0.4 (80606, Ibidi, Munich). The next day medium was substituted with FBS-free medium for at least additional 24 h prior to treatment with 1–10 ng/ml EGF. Following incubation with EGF, 1 mM 5-ALAwas added to the cell culture. At the desired time point, cells were washed twice with PBS (10010-015, Gibco^® Life technologies™, UK), fixed with 4% paraformaldehyde/PBS for 10 min, washed with PBS (4 × 5 min) and stored at 4 °C until staining. Cells were permeabilized for 5 min with 0.5% ice cold Triton-X-100/PBS, blocked for at least 20 min with 1% BSA, followed by 1 h incubation with the selected primary antibody, diluted in 1% BSA/PBS. After washing with 1% BSA/PBS (3 × 10 min), cells were incubated with the appropriate secondary antibody diluted 1:1000 (Alexa Fluor^® 488 and/or Alexa Fluor^® 594; Invitrogen), washed with 1% BSA/PBS (2 × 10 min) followed by PBS (1 × 10 min) and incubated with 1 μg/ml DAPI/methanol for 3 min and washed with PBS (3 × 10 min). Slides were examined with a Nikon ECLIPSE Ti-E confocal microscope and a Nikon C2 camera. Merged z-stacks (z = 0.49 µm) were analyzed with NIS-Elements AR software (Nikon). At least 50 cells/condition were analyzed.

Extirpated LNs were snap frozen using dry ice in OCT media (Tissue-Tek) and kept in −80°C until use. Tissues were thawed to −20°C and then sectioned (8 µm) and fixed for 15 min in 2% formaldehyde in PBS. Tissues were permeabilized using tris-buffered saline with 0.1% saponin and 1% hepes buffer (permwash with pH 7.4), all future reagents were diluted in permwash. LNs were blocked with 1% FCS and then stained with anti CD3 (Dako), Ki67 (BD), and PD-1 (R&D systems). After this, biotinylated anti-mouse or anti-goat (Dako) or anti-rabbit (Vector Labs) secondary antibodies were added, which were detected by the addition of streptavidin-conjugated Alexa Fluor 405/555/647 (Invitrogen). Image tiles of entire LNs were acquired using a Nikon Eclipse Ti-E confocal microscope. GCs were defined as dense follicular structures including CD3+PD-1+ cells (light zone) and Ki67+ cells (dark zone) (Figure 3A). Image analysis was done using CellProfiler software (Broad Institute Inc.) with in-house algorithms. Briefly, GCs were manually identified in the program to enable automatic enumeration of PD-1+ and Ki67+ cells within the individual GCs and the area of the GCs. PD-1+ cells were almost exclusively CD3+ and Ki67+ cells were mostly CD3− (GC B cells).

BMMΦs and RAW 264.7 cells were seeded in sterile 24-well plates at a concentration of 0.2 × 10⁶ cells/well and then treated with 30 µg/mL of Cy5.5-labeled iNPs (PLA-PEMA and PLA-PVA) for 1 hr. All treated wells were washed twice with PBS to remove excess iNPs and replenished with 500 µL of fresh sterile PBS. For fluorescence microscopy, cells were either visualized immediately or fixed with 4% paraformaldehyde prior to visualization with either an ECHO (San Diego, CA, USA) Revolve benchtop fluorescence microscope or Nikon (Tokyo, Japan) Eclipse Ti-E confocal microscope. For flow cytometry, cells were scraped using a blunt 1000 µL pipette tip followed by collection by centrifugation and stained for viability using DAPI dye. Flow cytometry was used to measure Cy5.5 signal on viable (DAPI⁻) cells.