Briefly, splenic NK cells were stained with CD3-PE-Cy7 (clone 145-C211, BD Biosciences, Franklin Lakes, NJ), NK1.1-PerCP-Cy5.5 (clone PK136, BD Biosciences), Ly49A-PE (clone A1, BD Biosciences), Ly49C/I-FITC (clone 5E6, BD Biosciences), Ly49D-BV510 (clone 4E5, BD Biosciences), and Ly49G2-APC (clone 4D11, BD Biosciences) to assess Ly49 receptor expression. To detect NKG2D expression, a separate cell preparation was stained with CD3, NK1.1, and NKG2D-APC (clone CX5, BioLegend). After isolation post-transplantation, allograft endothelial cells were stained with CD45-PerCP-Cy5.5 (clone 104, BD Biosciences), CD31-FITC (clone 390, Invitrogen), Rae-1-BV421 (clone 186107, BD Biosciences), H60-PE (clone 205326, R&D Systems (Minneapolis, MN)), and MULT-1-APC (clone 237104, R&D systems). To detect anti-donor MHC Class I antibody bonding, naïve endothelial cells were stained with CD45-PerCP-Cy5.5 (clone 104, BD Biosciences), CD31-PE (clone 390, BioLegend), and mouse IgG-FITC (clone poly 4060, BioLegend) after incubation with the appropriate monoclonal antibody. After staining, cells were washed twice with PBS with 1% bovine serum albumin (BSA) and 0.1% sodium azide; data was collected using a BD LSR II or BD Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (BD Biosciences, Version 10.6.2).
Cd45 percp cy5
Manufactured by BD
Sourced in United States, United Kingdom
CD45-PerCP-Cy5.5 is a fluorochrome-conjugated antibody that binds to the CD45 antigen, a cell surface glycoprotein expressed on all human leukocytes. This reagent is designed for use in flow cytometry applications to identify and enumerate various leukocyte populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BD (12 mentions)
Cd4 pe cy7, by BD (9 mentions)
Cd8 apc cy7, by BD (9 mentions)
Cd19 apc, by BD (8 mentions)
Multitest 6 color tbnk reagent, by BD (5 mentions)
Facsdiva software, by BD (4 mentions)
Cd4 fitc, by BD (4 mentions)
Cd56 pe, by BD (4 mentions)
Ly6g fitc, by BD (4 mentions)
Lsrii flow cytometer, by BD (4 mentions)
Facscanto 2, by BD (4 mentions)
Cd8 apc, by BD (3 mentions)
Facscanto, by BD (3 mentions)
Cd11b fitc, by BD (3 mentions)
Foxp3 apc, by Thermo Fisher Scientific (3 mentions)
Gr1 apc, by BD (3 mentions)
Cd11c pe, by BD (3 mentions)
Facscanto clinical software, by BD (3 mentions)
Cd11b apc, by BD (3 mentions)
Cd4 apc, by BD (3 mentions)
51 protocols using cd45 percp cy5
1
Comprehensive Immunophenotyping of Splenic NK Cells
2
Quantitative Analysis of Immune Cells
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Stained cells were analyzed using a BD LSR II-SORP or Fortessa system (Becton Dickinson) and analyzed with FlowJo software (Tree Star). The following anti-mouse mAbs were used: CD45- PerCPCy5, CD45-APC, CD8-APC, anti-IFNγ-PECy7, and anti-IL-4-FITC (BD Biosciences), CD4-AF700, CD11b APC-eFluor780, and c-kit PECy7 all from (eBiosciences). Anti human CD2 (BioLegend) was used to detect IL-4 production in KN2 mice. To detect IL-4Rα on primary keratinocytes, the following anti-mouse mAbs were used: CD124, anti IL-4Rα-PE (BD, Pharmingen), and CD49f-Brillant Violet 421 (BioLegend). Imaging flow cytometry was performed using an ImageStream cytometer (Amnis, Millipore) at low speed. Data were analyzed with the IDEAS software.
3
Flow Cytometry Analysis of Immune Cells
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Stained cells were analyzed using a BD LSR II-SORP system (Becton Dickinson) and analyzed with FlowJo software (Tree Star). The following mAbs were used: CD45-PerCPCy5, CD45-APC, CD8-APC, anti-IFNγ-PECy7 and anti-IL-4-FITC (BD Biosciences), Gr1-APC, CD11c-PECy7, CD11b-PB, F4/80-APC (eBiosciences), 1A8-APCCy7, Ly6C-FITC, 1A8-APCCy7, CD4-AF700. DAPI and AnV-FITC (BioLegends). In vivo bioluminescence imaging of MPO activity was quantified through injection of 200mg/kg luminol (Carbosynth) i.p. 10 minutes before luminescence acquisition. Photon emission was acquired for 10 minutes using a Xenogen IVIS Imaging system.
4
Progenitor Cell Characterization in BMC
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Flow cytometry was applied to determine the frequency of immature hematopoietic stem cells (CD34+/CD133+/CD45+), more mature progenitor cells (CD34+/CD133−/CD45+), and putative MSC-precursors (CD271+/CD45−) in the BMC preparations prior to the seeding procedure as well as in the supernatant after the seeding procedure to determine whether these cell types differ in their adhesive capacity to the scaffolds. Increased adhesion to the scaffold would result in a decreased percentage of that progenitor cell species in the fraction of nonadhering cells and a decreased adhesion to the scaffold would cause a relative increase of that progenitor cell species in the fraction of the nonadhering cells.
5 × 105 BMC were suspended in 100 μL PBS and were incubated for 30 minutes at 4°C with 10 μL of each CD34-FITC (BD-Biosciences, Heidelberg, Germany), CD133-PE (Miltenyi-Biotech, Bergisch-Gladbach, Germany), CD45-PerCP-Cy5 (BD-Biosciences), and CD271-APC (RnD-Systems, Wiesbaden, Germany). Fluorochrome conjugated isotype identical antibodies served as control. After incubation, the cells were washed twice and immediately analyzed by flow cytometry. 1 × 105 mononuclear cells were acquired based on their forward and side scatter properties.
5 × 105 BMC were suspended in 100 μL PBS and were incubated for 30 minutes at 4°C with 10 μL of each CD34-FITC (BD-Biosciences, Heidelberg, Germany), CD133-PE (Miltenyi-Biotech, Bergisch-Gladbach, Germany), CD45-PerCP-Cy5 (BD-Biosciences), and CD271-APC (RnD-Systems, Wiesbaden, Germany). Fluorochrome conjugated isotype identical antibodies served as control. After incubation, the cells were washed twice and immediately analyzed by flow cytometry. 1 × 105 mononuclear cells were acquired based on their forward and side scatter properties.
5
Cell Surface Marker Expression Profiling
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We analyzed cell surface expression with a pre-defined set of protein markers. These
assays were performed using commercially available monoclonal antibodies, following the
manufacturers’ instructions. Briefly, the cells at third passage were harvested by a
treatment with 0.25% Tryple Express (Gibco-Invitrogen, Carlsbad, CA, USA), washed with PBS
(pH = 7.4) and stained with the selected monoclonal antibodies and incubated in the dark
for 30 min at 4°C. Cells were then washed and fixed with 1% paraformaldehyde. The
following human antibodies were used: CD14-FITC (clone: M5E2; BD Pharmingen, San Diego,
CA, USA), CD29-PE (clone: MAR4; BD Pharmingen), CD31-PE (clone: WM59; BD Pharmingen),
CD34-PE (clone: 581; BD Pharmingen), CD44-PE (clone: 515; BD Pharmingen), CD45-PerCPCy5
(clone: 2D1; BD Biosciences, San Jose, CA, USA), CD73-PE (clone: AD2; BD Pharmingen),
CD90-APC (clone: 5E10; BD Pharmingen), CD106-FITC (clone: 51-10C9; BD Pharmingen),
CD166-PE (clone: 3A6; BD Pharmingen), HLA-DR-PerCP-Cy5 (clone: L243; BD Biosciences), and
CD105-PE (clone: 8E11; Chemicon, Temecula, CA, USA). Cells were analyzed using FACSARIA
flow cytometry equipment (BD Biosciences) and data analyses were performed using FACSDIVA
software (BD Biosciences) or Flow Jo Software (TreeStar, Ashland, OR, USA).
assays were performed using commercially available monoclonal antibodies, following the
manufacturers’ instructions. Briefly, the cells at third passage were harvested by a
treatment with 0.25% Tryple Express (Gibco-Invitrogen, Carlsbad, CA, USA), washed with PBS
(pH = 7.4) and stained with the selected monoclonal antibodies and incubated in the dark
for 30 min at 4°C. Cells were then washed and fixed with 1% paraformaldehyde. The
following human antibodies were used: CD14-FITC (clone: M5E2; BD Pharmingen, San Diego,
CA, USA), CD29-PE (clone: MAR4; BD Pharmingen), CD31-PE (clone: WM59; BD Pharmingen),
CD34-PE (clone: 581; BD Pharmingen), CD44-PE (clone: 515; BD Pharmingen), CD45-PerCPCy5
(clone: 2D1; BD Biosciences, San Jose, CA, USA), CD73-PE (clone: AD2; BD Pharmingen),
CD90-APC (clone: 5E10; BD Pharmingen), CD106-FITC (clone: 51-10C9; BD Pharmingen),
CD166-PE (clone: 3A6; BD Pharmingen), HLA-DR-PerCP-Cy5 (clone: L243; BD Biosciences), and
CD105-PE (clone: 8E11; Chemicon, Temecula, CA, USA). Cells were analyzed using FACSARIA
flow cytometry equipment (BD Biosciences) and data analyses were performed using FACSDIVA
software (BD Biosciences) or Flow Jo Software (TreeStar, Ashland, OR, USA).
6
Immunophenotypic Characterization of ADSCs
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ADSCs and IGF-1-ADSCs were subjected to immunophenotypic characterization. Cultured ADSCs were harvested with 0.25% trypsin and suspended as single cells at a concentration of 5 × 10 5 cells in 500 µl PBS (10 mM, pH 7.4). To verify that cultured cells were MSCs, the cells were analyzed by flow cytometry (FACSCanto; BD Biosciences, Franklin Lakes, NJ, USA) after labeling with CD45-PercP-CY5 (BD Pharmingen™, San Diego, CA, USA), CD34-PE (Santa Cruz Biotechnology, Dallas, TX, USA), CD11b-FITC (Caltag Laboratories, Burlingame, CA, USA), CD29-FITC (BD Pharmingen), and CD90-FITC (Caltag Laboratories). Isotype IgG1 immunoglobulin served as a control. In addition, these cells were cultured in the presence of osteogenic medium [10 nM dexamethasone, 10 mM b-glycerophosphate, 50 µg/ml l-ascorbate 2-phospate, and 10 nM 1a,25-dihydroxyvitamin D 3 (all from BIOMOL Research Laboratories Inc., Plymouth Meeting, PA, USA)] and adipogenic medium [DMEM-low glucose supplemented with 15% fetal calf serum (FCS), 50 U/ml penicillin, 50 mg/ml streptomycin, 5 × 10 -4 M indomethacin, 10 µg/ml human insulin, and 10 -7 M dexamethasone (all from Sigma-Aldrich)].
7
Comprehensive Immune Cell Profiling
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Monoclonal antibodies CD3e-ApcCy7, CD45-PerCPCy5.5, Gr1-APC, CD8-PECy7, CD25-FITC, CD11b-FITC, CD11C-PE, B220-PB, and NK1.1-PE were purchased from BD Biosciences. Foxp3-APC and F4/80-PE were purchased from eBioscience and CD4-PO and calcein violet from Invitrogen.
8
Profiling HIV Reservoir Dynamics
9
Comprehensive Immune Cell Profiling
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The proportions of neutrophils, lymphocytes, monocytes, CD4+ T, CD8+ T, CD19+ B, NK (CD3-CD56+), and NKT (CD3+CD56+) cell were analyzed by flow cytometry. The absolute counts of immune cells were measured by using BD TruCount Absolute-Count Tubes (BD340334). The following antibodies (CD3-FITC, CD8-APC-Cy7, CD4-PE-Cy7, CD45-Percp-Cy5.5, CD56-PE, and CD19-APC) were used, and all reagents were purchased from BD Biosciences. All samples were detected by a BD FACS Canto II flow cytometry system and analyzed with the BD FACS Diva software.
10
Multiparametric Flow Cytometry for Lymphocyte Profiling
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At the same time as serological testing, whole blood draws for flow cytometry analysis (3 mL/subject) were collected in ethylenediaminetetraacetic acid (EDTA) tubes. The BD Multitest 6-color TBNK reagent was used to determine absolute counts of B and NK, as well as CD4 and CD8 subpopulations of T cells. The panel for staining included the following monoclonal antibodies: CD3 FITC, CD4 PE-Cy7, CD8 APC-Cy7, CD19 APC, CD45 PerCP-Cy5.5, and CD16 PE + CD56 PE; BD Biosciences, San Jose, CA). The BD Trucount tubes (BD Biosciences, San Jose, CA) were filled with 20 µL of BD Multitest 6-color TBNK reagent and 50 µL aliquots of EDTA-anticoagulated whole blood. The mixture was incubated at room temperature in the dark for 20 min before being lysed with 2 mL of FACS Lysis Solution (BD Biosciences, San Jose, CA). After an additional 15 min of incubation, the erythrocyte-lysed, unwashed, and stained samples were analyzed. The data were acquired using the BD FACSCanto II system and BD FACSCanto clinical software (BD Biosciences, San Jose, CA). The calibration of the instrument with BD FACS 7-color setup beads was confirmed before each running process according to the manufacturer’s instructions [16] . The results for each lymphocyte subset were reported as absolute cell counts/µL.
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