Bovine serum albumin (bsa)

Cells were processed using the protocol described by Rothkamm et al. (17 (link)). Briefly, cells were grown on sterile glass coverslips inside a 4-well plate at a density of 10⁴/mL. After 48 h, cells were irradiated with a desired dose (i.e., 20 mG or 2 Gy) at ~60–70% confluency alongside sham-irradiated controls. Cells were analyzed for γH2AX foci at 30 min, 4 and 24 h post-exposure. At these time points, cells were fixed for 5 min in phosphate buffered saline (PBS) with formaldehyde (4%), and permeabilized with Triton X-100 (1%) in PBS for 10 min. Subsequently, cells were blocked with bovine serum albumin (1%) for 30 min and immuno-stained for γH2AX (mouse anti-H2AX, 1:500 dilution, BioLegend^®, London, UK) at room temperature for 1 h. Secondary antibody (goat anti-mouse conjugated with Alexa Fluor^® 555, 1:500 dilution, Fisher Scientific™, Loughborough, UK) and DAPI (1:500) were then added and incubated for 30 min at room temperature. Cells were eventually washed (3x5 min) in PBS to remove excess secondary antibodies before mounting with Vectashield Vibrance^® (Vector Laboratories, Kirtlington, UK). Slides were imaged using Nikon Ti-Eclipse fluorescent microscope equipped with NIS-Elements AR software (version 413.05). One thousand foci or 1,000 cells were scored for each condition, whichever came first based on power and sample size calculations.

Cells were fixed with 4% paraformaldehyde (Biolegend, USA) for 10 min and permeabilized with 0.1% Triton X100 (Merck KGaA, Germany) for 10 min. Cells were washed three times with PBS after each step. Afterwards, cells were stained with Phalloidin conjugated with Alexa Fluor 594 (dilution 1:250 in PBS; Invitrogen, Carlsbad, CA, USA) and Hoechst-33342 (dilution 1:10,000 in PBS; Invitrogen, Carlsbad, CA, USA). For staining the αSMA, cells were blocked with 1% bovine serum albumin for 1 h at room temperature, incubated with mouse anti-human αSMA (dilution 1:250 in PBS; Biolegend, USA) overnight at 4 °C, and incubated with goat anti-mouse IgG conjugated with Alexa Fluor-488 (dilution 1:250 in PBS; Invitrogen, USA) for 2 h. Cells were washed three times with PBS after each step. Cell imaging was performed using an epi-fluorescence microscope (DMi8 S; Leica, Germany) using a ×20 long distance objective (NA 0.4; Leica, Germany).

After enrichment for A549TT cells, recovered samples were incubated in phosphate-buffered saline (FUJIFILM Wako, Osaka, Japan) containing 1% bovine serum albumin (Lampire, Pipersville, USA) for 20 min and centrifuged at 500 × g for 10 min. Samples were then incubated with 100 μL of antibody solution (Alexa Fluor 647-labeled anti-cytokeratin antibody [Clone C11; Cell Signaling Technologies, Danvers, USA]) (1:20) and PE-Cyanin 7-labeled anti-CD45 antibody (Clone HI-30; BioLegend, San Diego, USA) (1:50) containing 0.1% HOECHST33342 and 1% bovine serum albumin in phosphate-buffered saline for 60 min at room temperature. Cytokeratin was used as a marker for spiked cells, and CD45 was used as a marker of WBCs.

MSCs were fixed with 4% paraformaldehyde solution (Panreac) at room temperature for 10 min and incubated with 0.2% Triton ×100 (Sigma, St. Louis, MO, USA) solution at RT for 10 min (except neurotrimin labelling). Furthermore, MSCs were incubated for 1 h in 1% bovine serum albumin (BSA, Sigma) and 10% normal goat serum (Abcam, Cambridge, UK) solution at room temperature to block the non-specific interaction of antibodies. Subsequently, the samples were incubated with primary polyclonal rabbit antibody for αSMA (Biolegend, San Diego, CA, USA, 904601), perilipin (Thermo Fisher Scientific, PA1-1051), CHD3 (Cloud-Clone Corp., Wuhan, China, PAA317Mu01), neurotrimin (Affinity Biosciences, Melbourne, Victoria, Australia, DF4245), RDH10 (Affinity Biosciences, DF12105), or rabbit polyclonal IgG (Biolegend, 910801) in 1% BSA solution at +4° overnight. Then, the samples were incubated with fluorescence-labeled goat anti-rabbit or goat anti-mouse (Invitrogen, A-11001) secondary antibodies (A11034, Invitrogen) at room temperature for 1 h. Cell nuclei were labeled with DAPI (DAKO, Glostrup, Denmark). Samples were analyzed with a Leica DM6000B fluorescent microscope equipped with a Leica DFC 360FX camera (Leica Microsystems GmbH, Wetzlar, Germany) using the LasX program. The percentage of CHD3+ MSCs was evaluated in FIJI using IgG-based thresholding.

The keratinocytes co-cultured with M. sympodialis, MalaEx or LPS, or cultured alone in the 8 well μ-slides with a glass coverslip bottom were directly fixed in 4% formaldehyde for 10 min, treated with 0.5% Triton-X-100 (BDH Laboratory Supplies, Poole, UK) for 5 min, and blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich) for 5 min at RT. Thereafter, Alexa Fluor 488 mouse anti-CD54/ICAM-1 or Alexa Fluor 488 IgG₁ isotype control (Biolegend, San Diego, CA, USA), both at 1:20 dilution in 5% BSA, were added for 1 h at RT. The glass coverslip bottoms were finally covered with Prolong Gold antifade mountant (Invitrogen, Thermo Fisher Scientific, MA, USA). Fluorescent images as z-scans and phase contrast images were acquired on a CLSM (TCS SP2; Leica Microsystems, Mannheim, Germany). Each florescent image was combined with each corresponding phase contrast image. Confocal images were used to manually calculate the % of ICAM-1 positive keratinocytes defined as strongly positive. For each culture condition 100 cells were analyzed.