αcd45 pe

After a four-day treatment, Lipogems-treated and control cells were characterized by flow cytometry for the expression of key stem cell markers. Flow cytometry analysis was performed on 1 × 10⁵ cells/sample. Briefly, aspecific binding sites were blocked with a blocking solution (50% 1x PBS, 50% FBS) for 30 min at room temperature and washed twice with PBS at 4°C. Cells were stained with fluorochrome-conjugated mouse antihuman antibodies at the optimal concentration (1 : 20 dilution) in PBS for 10 min at 4°C and washed twice with PBS at 4°C. Cell characterization was performed using the following antibodies: αCD9 FITC, αCD73 FITC, αHLA-DR FITC, αCD13 PE, αCD29 PE, αCD44 PE, αCD45 PE, αCD71 PE, αCD90 PE, αCD105 PE, αCD106 PE, αCD34 PerCP-eFluor710, αCD166 PerCP-eFluor710, αHLA-ABC FITC, αNG2 PE, and αSSEA-4 PE (all from eBioscience); αLineage Cocktail FITC, αCD18 PE, αCD140a PE, αCD140b APC, αCD146 PE, and αStro-1 Alexa Fluor 647 (all from BioLegend); and αCD117 PE (Miltenyi Biotec). The respective isotype antibodies were used as controls. Samples were acquired with a Navios Flow Cytometer (Beckman Coulter) and data were processed with Kaluza software (Beckman Coulter).

αCD2-biotin (clone: RPA-2.10), αCD14-biotin (clone: HCD14), αCD16-biotin (clone: 3G8), αCD19-biotin (clone: 561), αCD235a-biotin (clone: HIR2), αCD3-biotin (clone: OKT3a), αCD34-APC (clone: 561), αCD38-PE/Cy7 (clone: HIT2), αCD4-APC/Cy7 (clone: RPA-T4), αCD8a-Pacific Blue (clone: RPA-T8), αCD90-PerCP/Cy5.5 (clone: 5E10), αCD272-FITC (αBTLA; clone: MIH26), αIgG2-FITC (clone: MOPC-173), αCD200R-PE (clone: OX-108), αIgG1-PE (clone: MOPC-21), αCD160-PerCP/Cy5.5 (clone: BY55), αIgM-PerCP/Cy5.5 (clone: MM-30), αCD96-APC (clone: NK92.39), αIgG1-APC (MOPC-21), αCD28-PE/Cy7 (clone: CD28.2), αIgG1-PE/Cy7 (MOPC-21) and αStreptavidin-FITC were from BioLegend, USA. αCD45-PE (clone: HI30) was from eBioscience, USA. αIgG1-PE (clone: IS11-12E4.23.20) was from Miltenyi Biotec, Germany. Samples were analyzed on BD LSRFortessa™ (BD Biosciences, USA). Data was analyzed using FlowJo software v.10.7 (FlowJo, LLC, USA).

BALs were collected using 3 ml of PBS and a further 5 ml of PBS were added at the time of preparation of the samples for FACS analysis. Cell pellets were resuspended in 1 ml red blood lysis buffer (Sigma). Blocking of the Fc receptor to remove unspecific signal was achieved by incubating the samples with 0.5 µg of an anti-Mouse CD16/CD32 antibody (E-bioscience) in 100 µl of 0.1% BSA PBS. 14 µl of antibody mix was added for labelling of macrophages (α-F4/80-APC-Cy7, 5 µl, Biolegend), leukocytes (α-CD45-PE, 2 µl, E-bioscience) and neutrophils (α-Ly-6G-BV421, 2 µl, Biolegend). Samples was analysed using a BD Fortessa cell analyser. Data acquisition and analysis were performed using respectively the software Diva and FlowJo. For each sample (n  =  4, plus 2 controls), cell population size for macrophages (F4/80⁺) and neutrophils (Ly-6G⁺) were expressed as cells/ml.