Mon162

Epidermal punch biopsies (6 mm diameter) in triplicates were floated on medium containing either a mouse α-RTN1A ab (mon162, abcam) or the respective isotype control ab (IgG1, abcam) (5 µg/ml/each) in 96-well round bottom plates at 37°C, 5% CO₂. Sheets were collected at 3, 6, and 24 hr, fixed with acetone and stained with a secondary α-mouse cross-absorbed F(ab’) ab fragment conjugated with AF546 (Thermo Fisher Scientific) and a FITC-labeled α-CD207 (Dendritics) to identify LCs. In some experiments, 24 hr cultured epidermal sheets were stained with a FITC-labeled α-vimentin, primary rat α-CD207 (Sigma-Aldrich) and mouse α-RTN1A abs (mon162, abcam) ON at 4°C, followed by α-mouse and α-rat secondary abs and counterstaining with DAPI. 3D projections have been created with ImarisViewer (v.9.8). The contrast and brightness of representative images (Figure 1B–C) remained unaltered, to highlight the detection level of the abs taken up by rLCs. All abs and reagents used in this study are listed in Key resources table.

Epidermal punch biopsies (6 mm diameter) in triplicates for each condition were floated on RPMI medium supplemented with 10% fetal bovine serum (FBS, Gibco), 1% P/S, and human TLR agonists (InvitroGen) in 96-well round bottom plates. The following TLR agonists were used: TLR1/2 (Pam3CSK4x3 HCl; 1 µg/ml), TLR2 (heat-killed L. monocytogenes; 10⁸ cells/ml), TLR3/7 ((poly(A:U)); 25 µg/ml), TLR3 (LMW and HMW poly(I:C); both at 0.8 µg/ml), TLR6/2 (mycoplasma salivarium, FSL-1, Pam2CGDPKHPKSF; 2.5 µg/ml) and TLR7 (imiquimod; 2.5 µg/ml). After 24 and 48 hr, epidermal sheets were collected, fixed with acetone (Merck) for 10 min at room temperature (RT), incubated with FITC-conjugated α-CD83 and α-CD86 abs, as well as with a primary mouse α-RTN1A ab (mon162, abcam) ON at 4°C. All abs were diluted in 2% bovine serum albumin (Gibco, Thermo Fisher Scientific) in PBS. Subsequently, sheets were incubated with an AF647-labeled α-mouse secondary ab (Thermo Fisher Scientific) for 1 hr at RT and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 1 min. After PBS washing, sheets were mounted (ProLong Gold antifade reagent, Invitrogen) with the stratum corneum facing the slide and imaged. At the same time points cultivation medium was harvested and stored at –80°C for further analyses and eLCs were processed and analyzed as described below.