Ix83 spinning disk confocal microscope

Immunocytochemical staining of MSC for expression of antimicrobial peptides was performed as described previously (9 (link), 16 (link)). Briefly, 1 X 10⁴ cells were seeded on round coverslips (Chemglass Life Sciences, Vineland, NJ) placed within 24-well-cell culture plates overnight, then fixed with 4% paraformaldehyde (Fisher Scientific, Hampton, New Hampshire) for 10 min, washed with PBS and permeabilized with 0.1% Triton X-100(Sigma-Aldrich,). Slides were then blocked using 5% v/v normal donkey serum and then incubated with primary antibodies, diluted appropriately. Antibodies used for these studies included surfactant protein D antibody (ab203309), lipocalin-2 antibody (ab63929), beta 2 defensin antibody (ab9871), hepcidin antibody (ab134790), and cathelicidin antibody (ab180760), all obtained from Abcam (Cambridge, MA). Specificity controls for immunostaining included purified IgG antibodies from non-immune rabbits or goats. Following primary antibody incubation, slides were washed and incubated with secondary antibodies, either donkey anti-rabbit or donkey anti-goat, both conjugated to Cy3 (Jackson ImmunoResearch Laboratories, Inc, West Grove, Pennsylvania) and the slides were then counter-stained with DAPI to visualize cell nuclei. Image capture for fluorescence staining was done using an Olympus IX83 spinning disk confocal microscope.

ICC was performed according to previously published protocol (28 (link)). Primary antibodies used in these studies include: anti-vimentin (clone V9; Merck Millipore, Billerica, MA), anti-TUJ1 (beta III tubulin; clone TUJ1 Covance Inc. San Diego, CA), and anti-MAP2α (clone AP20; Merck Millipore, Billerica, MA). Corresponding rabbit and mouse irrelevant isotype antibodies were used at concentrations matching the primary antibodies (eBioscience Inc, San Diego CA). Secondary antibodies used were donkey anti mouse IgG or donkey anti rabbit IgG (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) conjugated to Cy3. Visualization of florescence staining was performed on Olympus IX83 spinning disk confocal microscope. Images were imported as Tiff files to Photoshop CC 2015, and adjusted with high definition resolution (HDR) toning. For each stain, HDR toning was applied to corresponding isotype control stains as well.

In all, 500 000 human neutrophils were plated on 0.01% poly‐L lysine (Sigma‐Aldrich) coated coverslips (Chemglass Life Sciences LLC) in 24‐well cell culture plates, then incubated with 0.5 mL MSC CM for 3 hours. Studies were done using neutrophils obtained from three unrelated, healthy donors. IRB approval was obtained for collection of human blood samples with signed informed consent. After washing off the CM, S. aureus was added at an MOI of 1 for the indicated time points in HBSS containing calcium, magnesium and 10% autologous human serum. Staining for neutrophil extracellular trap (NET) formation was performed according to a published protocol.28, 30 Cells were immunostained with antibodies for detection of histone H3 using Anti Histone H3 (RM188) Antibody (Caymen Chemical, Ann Arbor, Michigan) and neutrophil elastase Anti‐Neutrophil Elastase antibody (ab21595) (Abcam) with slight modifications for staining in a 24‐well plate. Images were taken on Olympus IX83 spinning disk confocal microscope, by imaging 15 random fields per condition. The NET area was calculated using ImageJ31; and the NET area (in pixels) was determined by merging channel 2 and 3 pixels (representing histone H3 and neutrophil elastase staining, respectively) then normalized to DAPI channel area.