Lsrfortessa hts flow cytometer

Wound edge skin at specific time points was collected and full-thickness skin samples were minced with surgical scissors and incubated for 30 min in dispase I solution (Roche, Basel, Switzerland) (2.4 μg/mL) at 37 °C. This was followed by incubation at 37 °C for 3 h with 2 mg/ml Collagenase D (Roche) at 37 °C under constant agitation. Obtained single-cell suspensions were washed with IMEM (Gibco-Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 0.15% sodium hydrogencarbonate, 1 mM sodium pyruvate, nonessential amino acids, and 50 μg/ml gentamycin. Single-cell suspensions were incubated with combinations of fluorophore-conjugated antibodies against the following surface markers: CD45-APCCy7 (30-F11), CD3e-BV711(17A2), F4/80-PE (BM8), CD11b-PE Dazzle 594 (M1/70), Ly6C-PerCP (HK1.4), Ly6G-BUV395 (1A8) in Hank’s buffered salt solution (HBSS) for 30 min at 4 °C and then washed. LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen Life Technologies) was used to exclude dead cells. Cells were then fixed for 30 min at 4 °C using BD Cytofix/Cytoperm (Becton Dickinson) and washed twice. All antibodies were purchased from Biolegend and BD Biosciences. Cell acquisition was performed on a BD LSR-Fortessa–HTS flow cytometer using FACSDiVa software (BD Biosciences) and data were analyzed using FlowJo software (TreeStar).

Protein aggregates in CLB-Ga and CLB-Sedp cell lines treated for 24 h with 10 µM or 10 nM bortezomib were analyzed using the proteostat detection kit (ENZ-51035-K100) according to the manufacturer’s intructions. Briefly, after treatment, cells were harvested and washed in PBS. Cells were then fixed in 4% PFA for 30 min at room temperature. Cells were then washed in PBS and permeabilized using 0.5% Triton in PBS for 30 min on ice. Cells were washed in PBS and incubated for 30 min with the Aggresome Red Detection Reagent (1:10,000). Cell fluorescence was analyzed using the LSR Fortessa HTS flow cytometer (BD Biosciences).