Surface receptors on live cells were stained with fluorochrome-conjugated antibodies described in Table S2 . Parallel detection of mitochondrial mass and potential was carried out by incubating surface-stained cells with fluorescent molecular probes (see below). Intracellular antigens, such as mTOR substrates pS6RP and pAkt, and transcription factors FoxP3 and Helios, were detected following permeabilization and fixation using FixPerm kit (eBioscience Cat Nos 00-5123-43, 005223-56, 008333-56). In agreement with a recent study123 (link), we have noted far greater sensitivity and accuracy and less variability of flow cytometry as compared to western blot detection of mTORC1 and mTORC2 activities13 (link),15 (link),100 (link)–102 (link). Intracellular cytokine production was assessed following 3 h stimulation with phorbol 12-myristate 13-acetate (PMA, 5 ng/ml; Sigma, cat. no. P-8139) and ionomycin (500 ng/ml; Sigma, cat. no. I-0634) in the presence of brefeldin A (10 ng/ml, Sigma cat no. B6542). Subsequently, cells were i) stained with antibodies directed to surface antigens; ii) fixed in 1% paraformaldehyde; iii) permeabilized with the eBioscience FixPerm kit; and iv) stained with antibodies directed to cytokines (Table S2 ).
Fix perm kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fix/Perm kit is a laboratory consumable product designed for the fixation and permeabilization of cells. It provides a standardized solution for the preparation of samples prior to further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (6 mentions)
Facscanto 2, by BD (4 mentions)
Lsrii flow cytometer, by BD (4 mentions)
Lsrfortessa flow cytometer, by BD (4 mentions)
Ionomycin, by Merck Group (3 mentions)
Facsaria 3, by BD (3 mentions)
Facscanto 2 cell analyzer, by BD (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Lsrfortessa, by BD (3 mentions)
Facsdiva, by BD (3 mentions)
Efluor 450, by Thermo Fisher Scientific (3 mentions)
Brefeldin a, by BioLegend (3 mentions)
Golgistop, by BD (3 mentions)
Cytexpert acquisition and analysis software, by Beckman Coulter (3 mentions)
Cytoflex cytometer, by Beckman Coulter (3 mentions)
Apc cy7 conjugated anti cd45, by BD (2 mentions)
Percp cy5.5 conjugated anti cd105, by BD (2 mentions)
Pe conjugated anti cd34, by Miltenyi Biotec (2 mentions)
Percp cy 5.5 conjugated anti nanog, by BD (2 mentions)
Accuri c6, by BD (2 mentions)
93 protocols using fix perm kit
1
Multiparametric Flow Cytometry Analysis
2
Characterization of hDPSCs by Flow Cytometry
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Flow cytometry analyses were performed on hDPSCs at first passage of culture. Cells were incubated with FITC-conjugated anti-CD90, PerCP-Cy5.5-conjugated anti-CD105, APC-Cy7-conjugated anti-CD45 (all purchased from BD Pharmingen), and PE-conjugated anti-CD34 (Miltenyi Biotech) for phenotypic characterization, and FITC-conjugated anti-bone sialo-protein (BSP) (Biorbyt), anti-CFS-conjugated anti-osteopontin (OPN) (R&D Systems) and PerCP-Cy 5.5-conjugated anti-NANOG (BD Pharmingen) for evaluation of osteogenic differentiation and mesenchymal stemness. As negative controls, cells were stained with an isotype control antibody. After incubation with the antibody, cells were re-suspended in PBS and analysed using an FACS ARIA III or BD Accuri C6 (BD Biosciences). Human DPSCs were then sorted for CD34- and CD90-positive markers. The purity of sorting was approximately 90%.
For intracellular staining of osteocalcin (OC), OPN and NANOG, cells were processed using a Fix & Perm Kit (Invitrogen) following the manufacturer's guidelines. All data were analysed using FCS express version 3 (De Novo Software).
For intracellular staining of osteocalcin (OC), OPN and NANOG, cells were processed using a Fix & Perm Kit (Invitrogen) following the manufacturer's guidelines. All data were analysed using FCS express version 3 (De Novo Software).
3
Galectin-3 Expression in Adherent Cells
4
Evaluating Hematopoietic Stem Cell Markers
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Flow cytometry analyses were performed on hDPSCs cultured in medium supplemented with C-FBS or NZ-FBS at first passage of culture. Cells were incubated with FITC-conjugated anti-CD90, PerCP-Cy5.5-conjugated anti-CD105, APC-Cy7-conjugated anti-CD45 (all purchased from BD Pharmingen, San Diego, CA), and PE-conjugated anti-CD34 (Miltenyi Biotech, Calderara di Reno, Bologna, Italy) for phenotypic characterization; and anti-Bone sialoprotein (BSP) (Abcam, Cambridge, UK), anti-CFS-conjugated anti-Osteopontin (OPN), PE-conjugated anti-Osteocalcin (OC) (both from R&D Systems, Minneapolis, MN) and PerCP-Cy 5.5-conjugated anti-Nanog (BD Pharmingen, Milan, Italy) to evaluate osteogenic differentiation and mesenchymal stemness. hDPSCs were sorted by CD34 expression. The purity of sorting was 90%. As negative controls, cells were stained with an isotype control antibody. After incubation with the antibody, cells were resuspended in PBS and analyzed with a FACS ARIA III (BD Biosciences, San Jose, CA). For intracellular staining of Osteocalcin, Osteopontin and Nanog, cells were processed using Fix & Perm Kit (Invitrogen, Milan, Italy) following the manufacturer's guidelines. All data were analyzed using FCS express version 3 (De Novo Software, Glendale, CA).
5
Activation and Phenotyping of γδ T Cells
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PBMC were cultured in RPMI 1640 supplemented with 10% FBS and 40μg/ml gentamycin. Stimulation was performed with 1μl (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP, Sigma). After 18 h of activation, Brefeldin A (GolgiPlug, BD Biosciences) was added. Five hours later, cells were stained with CD3-PerCP (OKT3), CD11c-PE-Cy7 (B-ly6), HLA-DR-PerCP-Cy5.5 (G46-6), CD69-Horizon PE-CF594 (FN50) (BD Biosciences) and γδTCR-PE (11F2) (Miltenyi Biotec). After surface staining cells were fixed and permeabilized with Fix/Perm Kit and intracellularly stained with IFN-γ-Alexa700 (B27) (Invitrogen). Data were acquired and analyzed as described for blood.
6
Phenotyping PBMC Subsets by Flow Cytometry
7
Quantification of T-cell Subsets in Spinal Cord
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On day 15, spinal cord tissue was extracted from euthanized mice and pooled for each treatment group (N = 6). Tissue was dissociated and lymphocytes collected at the interface of a 63% - 27% Percoll gradient, plated in Iscove’s Modified Dulbecco’s Medium (IMDM) and stimulated overnight with phorbol 12-myristate 13-acetate (20 nM and ionomycin (1 μM) in IMDM at 37°C. Brefeldin-A was added at a final concentration of 9 μg/ml and incubated for 6 hours. Fc receptors were blocked with 2.4 g2 Fc blocking reagent in 10% normal mouse serum, followed by extracellular staining with anti-CD4+ antibody conjugated to PE. Cells were then washed, fixed, and permeabilized (Fix & Perm kit, Invitrogen, Grand Island, NY) and stained with antibodies to Th1, Th2, and Th17 using FITC conjugated anti-IFN- γ, APC conjugated anti-IL-2, and PerCP Cy5.5 conjugated anti-IL-17, respectively (eBioscience, San Diego, CA) following modifications of published and manufacturer’s instructions [5 (link),6 (link),43 (link)]. Replicate samples of stained cells were processed in the Core Faculty using a FACSCalibur flow cytometer (BD Bioscience).
8
Leukocyte Phenotyping in Rat Blood
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Blood was collected into lithium-heparin microvette tubes from tail bleeds. Red blood cells were lysed (155 mM NH4Cl, 12 mM NaHCO3, 100 mM EDTA) and leukocytes washed in PBS before permeabilisation (fix-Perm kit, Invitrogen) and incubation with antibodies against rat Gran-PE (HIS48), MHC-II-perCP (HIS19) and CD68-Alexa Fluor 700 (ED1) (all 1:50; BioLegend). Cells were analysed by flow cytometry using an AccuriC6 (Becton Dickinson) with appropriate colour compensation and gates set to unstained cells.
9
Chondrocyte Type II Collagen Expression
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Chondrocytes, treated with BC (1% w/v), CS (1% w/v), and untreated (CTR) samples (at a density of 1 × 105 cells/sample) at the second passage of culture, were incubated with fluorescent‐labeled monoclonal antibody or respective isotype controls. The antibody used was anti‐collagen type II (COL2A1, AbCam). Secondary antibody was anti‐rabbit FITC (AbCam). For intracellular staining of COL2A1, cells were processed using the Fix & Perm Kit (Invitrogen) following the manufacturer's guidelines. The fluorescence associated to the cells was measured using FITC channel and calculated both as mean percentage of positivity for Type II collagen and as mean fluorescence intensity (MFI) for each sample. All data were acquired using FACS Aria III (BD) and analyzed using FCS version 3 software.
10
EGFR+ Cell Isolation and Analysis
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