Pe conjugated antibodies

For blood sampling at different time points (baseline, and at 18 h or on days 14 after induction of CLI), blood sampling from tail vein was performed using a 25# needle. After treatment with red blood cell-lysing buffer, the cells remained were labeled with appropriate antibodies. Flow cytometric analysis for identification of cell surface markers was performed based on our previous reports [50 (link)]. Briefly, the cells were incubated for 30 min with primary antibodies, including PE-conjugated antibodies (against CD34, Sca-1, CD31, BD Biosciences, San Jose, CA, USA), FITC-conjugated antibody against c-Kit (BD Biosciences), anti-CXCR4 (Abcam, Cambridge, MA, USA), and anti-KDR (NeoMarkers, Fremont, CA, USA) antibodies which were further recognized by Alexa flour 488-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Isotype-identical antibodies (IgG) served as controls. Flow cytometric analyses were performed by utilizing a fluorescence-activated cell sorter (Beckman Coulter FC500 flow cytometer, Brea, CA, USA).

Thymus and spleen tissue from control BALB/c mice and BALB/c-BALB/c Kaede chimeras was mechanically disrupted and stained with anti-CD4 BV711 (RM4-5; BioLegend), anti-CD8 BV786 (53–6.7; BioLegend), anti-TCRβ APC-Cy7 (H57-597; eBioscience), and PE-conjugated antibodies (all BD Biosciences) to TCRVβ3 (KJ25), TCRVβ5 (MR9-4), and TCRVβ11 (RR3-15). Data on percentages and numbers of TCRVβ⁺ cells from indicated tissues are shown after pregating on SP CD4⁺TCRβ⁺ cells or SP CD8⁺TCRβ⁺ cells as indicated.

Site densities of TCR, CD4, and pMHC on RBCs, Jurkat cells, THP-1 cells, glass beads, or origami beads were measured by flow cytometry using PE-conjugated antibodies from BD Biosciences (San Jose, CA): anti-human TCR-β1 (clone JOVI.1, 1:20), anti-human CD4 (clone OKT4, 1:20), and anti-human HLA-DR (clone L243, 1:5). Isotype control antibodies were PE mouse IgG2a κ (clone G155-178, 1:20) for TCR and HLA-DR, and PE mouse IgG2b, κ (Clone 27-35, 1:20) for CD4. RBCs or beads were incubated with corresponding antibodies in 1X PBS + 2% BSA for 30 min at 25 °C and washed three times before being analyzed using the BD Accuri or Fortessa Flow Cytometer. Data were collected with FACSDiva v9 and analyzed with Flowjo v10. An example of gating strategy is shown in Supplementary Figure 5. Site density was calculated based on antibody-conjugated PE fluorescence relative to the QuantiBRITE PE standard beads (BD Biosciences). For micropipette experiments, RBCs bearing protein of interest (TCR, CD4, or MHC) were prepared for flow cytometry using three separate samples: one saturating the RBC surface, one of the experimental conditions, and one isotype control. Brightness across all antibodies was normalized against the CD4 staining which was brighter than the TCR staining.

The quality of immature DCs was assessed by staining with anti-human CD83 and CD14 phycoerythrin (PE)-conjugated antibodies (BD). DCs were used if their purity was more than 95% and their expression of CD14 and CD83 was less than 10%. To evaluate the level of HSV-2 infection in immature DCs, cells were permeabilized with phosphate-buffered saline (PBS) containing 0.2% saponin and 0.2% FCS and incubated with polyclonal Ab (PAb) against HSV-2 (identifying all major glycoproteins in the viral envelope and at least one core protein; B0116; Dako, Denmark) or monoclonal Ab (MAb) against HSV-2-infected cell protein 8 (ICP8) (4E6) (Santa Cruz Biotechnology, USA), followed by fluorescein isothiocyanate (FITC)- or Alexa 488-conjugated secondary Ab (Abcam). A Zombie Aqua Fixable Viability kit (BioLegend) was used to discern viable/dead cells. Additionally, PE-conjugated MAb against CD11b/Mac-1 (BD) was used to evaluate the presence of CD11b receptor on DCs. The samples were assessed by flow cytometry (FACSCanto; BD) and analyzed with FlowJo (Treestar, Ashland, OR, USA).

Human ADSCs were characterized using flow cytometry analysis [10 (link)]. 100,000 cells were harvested and incubated with either FITC or PE-conjugated antibodies against human CD24, CD90, CD44, CD105, CD73, HLA-ABC, HLA-DR, CD34, and CD45 (BD Bioscience, Erembodegem, Belgium) and CD29 (Acris, Herford, Germany) mouse anti-human monoclonal antibodies and appropriate isotype controls. Stained cells were analyzed using a Beckton Dickinson flow cytometer (Beckton Dickinson, Franklin Lakes, NJ, USA) using the Cell Quest software and data were analysed using the FlowJo software (Tree Star, Ashland, OR, USA).
The same analysis was performed for rat ADSCs using either FITC or PE-conjugated antibodies against rat I-E[κ] CD90, CD44, CD31, CD45, and CD11b (BD Bioscience) and CD29 (Acris) mouse anti-rat monoclonal antibodies and appropriate isotype controls.

For phenotype analysis, third-generation LGMSCs were digested, harvested, and washed with cold PBS. The cells were then labeled with PE-conjugated antibodies against CD29, CD73, CD31, CD45 and isotype control IgG1 or labeled with PE-conjugated antibodies against CD80, CD86, HLA-DR and isotype control IgG1 and IgG2a (BD biosciences, Franklin Lake, NJ, USA) (for immunogenicity analysis) for 30 min in the dark at 4 ℃. The cells were washed twice with stain buffer and analyzed using flow cytometry.
To evaluate the multilineage differentiation capacity, LGMSCs were cultured in the corresponding differentiation medium after reaching 80% confluence. The osteogenic induction medium consisted of α-MEM supplemented with 10% FBS, 10 nM dexamethasone (Sigma-Aldrich, St Louis, MO, USA), 0.2 mM ascorbic acid (Sigma-Aldrich), and 10 mM β-glycerophosphate disodium salt hydrate (Sigma-Aldrich). The medium was refreshed every 3 days. After 3 weeks, alizarin red (Sigma-Aldrich) staining was used to detect the formation of mineralized nodules. The adipogenic induction medium consisted of α-MEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 2 μM dexamethasone, 0.2 mM indomethacin (Sigma-Aldrich), and 0.01 g/L insulin (Sigma-Aldrich). After 2 weeks, the cells were stained with oil red O (Sigma-Aldrich).

The immunophenotype of purified (un)stimulated NK cells was evaluated using PE-conjugated antibodies (BD Biosciences) and flow cytometry for the following surface markers that might have a stimulating or inhibiting effect on NK cell functions: NKG2D, DNAM-1, PD-1, TIM-3, LAG-3, NKp46, FasL, TRAIL and 2B4. In addition, corresponding ligands on the PCC and (primary) PSC were measured using PE-conjugated antibodies and flow cytometry for the following markers: MICA/B, ULBP1, ULBP2,5,6, ULBP3, ULBP4 (NKG2D ligands; antibodies all from R&D Systems), Galectin-9 (TIM-3 ligand; antibody from BD Biosciences), PD-L1 and PD-L2 (PD-1 ligands; antibodies both from BD Biosciences). Corresponding isotype controls were included for all samples and served as negative controls. All samples were measured on a FACSCan flow cytometer (BD Biosciences).