For the identification of CD4+ Th cell subsets and CD8+ Tc cells, we used cytoplasmic cytokine staining method. Briefly, whole blood were diluted to 1:1 with saline solution and incubated with phorbol-12-myristate 13-acetate (PMA) (25 ng/ml), ionomycin (1 μg/ml) and Golgi Stop brefeldin A (10 μg/ml) (all from Sigma Aldrich, St. Louis, Missouri, USA) for 5 h at 37°C in 5% CO2 milieu. The following monoclonal antibodies were used for cell surface staining: CD4-PE-Cy5 or CD8-PE-Cy5 (both from Beckman Coulter). The cells were then fixed and permeabilized with Intraprep™ permeabilization reagent (Beckman Coulter) according to the manufacturer’s instructions, and intracellular cytokines were stained with the combination of interferon (IFN)-γ-FITC, interleukin (IL)-4-PE, IL-10-PE (all from BD Biosciences) and IL-17-PE (R&D Systems, Minneapolis, MN, USA) monoclonal antibodies. Measurements were performed and data were analyzed on Coulter FC500 flow cytometer (Beckman Coulter) equipped with Kaluza 1.2a software. IgG1-FITC (BD Biosciences) and IgG1-PE (R&D Systems) isotype-matched antibodies were used during the identification. Cells were quantified as their percentage in the CD4+ or CD8+ lymphocyte population.
Igg1 fitc
Manufactured by BD
Sourced in United States, United Kingdom
IgG1-FITC is a fluorescently labeled immunoglobulin G1 (IgG1) antibody. It is used as a tool for the detection and visualization of target proteins or cells in various laboratory techniques, such as flow cytometry, immunohistochemistry, and immunocytochemistry.
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Lab products found in correlation
Igg1 pe, by BD (26 mentions)
Igg2a pe, by BD (7 mentions)
Igg1 apc, by BD (7 mentions)
Facscalibur, by BD (6 mentions)
Cd34 pe, by BD (6 mentions)
Cellquest software, by BD (6 mentions)
Igg2a fitc, by BD (5 mentions)
Cd45 fitc, by BD (5 mentions)
Facscan, by BD (4 mentions)
Cd73 pe, by BD (4 mentions)
Cd29 fitc, by BD (3 mentions)
Cd146 pe, by BD (3 mentions)
Igg1 pe cy5, by BD (3 mentions)
Cd3 fitc, by BD (3 mentions)
Hla dr fitc, by BD (3 mentions)
Cd14 pe, by BD (3 mentions)
Cd4 fitc, by BD (3 mentions)
Cd44 pe, by BD (3 mentions)
Accuri c6, by BD (3 mentions)
Ifn γ fitc, by BD (2 mentions)
55 protocols using igg1 fitc
1
Flow Cytometric Identification of Lymphocyte Subsets
2
Phenotyping rDFSCs and rDPCs by Flow Cytometry
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The phenotype of rDFSCs and rDPCs was identified by flow cytometric analysis. The MSC phenotyping cocktail comprised both positive (CD29-FITC, CD44/CD90-PE, BD Bioscience, USA) and negative (CD34-PE, CD45-FITC, BD Bioscience, USA) fluorochrome-conjugated monoclonal antibodies. IgG1-FITC and IgG1-PE (BD Bioscience, USA) were used as isotype controls. Third-passage rDFSCs and rDPCs were suspended to 5 × 105 cells/mL in PBS solution, stained with different antibodies for 30 min at 4 °C, washed with PBS, resuspended in FACS buffer, and analyzed using a MOFlo™ high-performance cell sorter (Beckman Coulter, USA).
3
Flow Cytometric Analysis of T-Cell Subsets
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Fresh peripheral venous blood was collected from all patients 1 day before biopsy. Anticoagulant (100 μL) was added to each of the two test tubes containing either 20 μL CD4-FITC (BD, Franklin Lakes, NJ, USA) or 20 μL CD8-PE (BD) and mixed evenly. Additionally, a test tube with blood from Control mice and IgG1-FITC (BD) or IgG1-PE (BD) monoclonal antibodies (20 μL) was also mixed. All test tubes were incubated at room temperature after mixing for 20 min; then, 2 mL of red cell lysate (BD Biosciences, San Jose, CA, USA) was added. After incubation at room temperature for 10 min, samples were centrifuged at 12,000 rpm for 5 min, the supernatant was discarded, and 2 mL of phosphate buffer pH 7.4 (0.05% PBS) was added. The samples were mixed well and washed 1–2 times. Cells were resuspended in 500 μL of PBS, and immediately tested by flow cytometry. We gated for small lymphocytes (R1 gate) based on the forward scatter and side scatter of the cells. Mouse IgG1-FITC and IgG1-PE monoclonal antibodies were used as isotype-negative controls.
The data were acquired using a FACScan flow cytometer (BD) and analyzed with CellQuest software (BD).
The data were acquired using a FACScan flow cytometer (BD) and analyzed with CellQuest software (BD).
4
Comprehensive Immune Cell Profiling
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Extracellular surface marker staining was performed with IgG1-FITC, IgG2a-FITC, IgG1-PE, αCD25-FITC, αCD40-PE, αCD69-PE, αCD70-PE, αCD80-FITC, αCD83-PE, αCD86-FITC, and PD-L1-PE (all from BD Biosciences, Heidelberg, Germany); IgG3-PE (eBioscience, Frankfurt, Germany); and αCCR7-FITC (R&D Systems, Minneapolis, MN, USA) for 30 min at 4 °C in PBS supplemented with 1% FCS and 0.02% sodium azide (Merck, Darmstadt, Germany). The cells were analyzed using a FACScan cytofluorometer equipped with CellQuest software (BD, Heidelberg, Germany). Analysis was performed with the FCS Express software (De Novo Software, Glendale, CA, USA). Specific MFIs were calculated by subtracting the background MFI obtained with the isotype controls.
5
Isolation and Stimulation of Splenic B Cells
6
T-Cell Maturation and Phenotyping
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Human CB T-cells, including those which were deficient in PKCζ, were maturated as previously described using phytohaemagglutinin (PHA) and IL-2 [22 (link),28 (link)]. Maturation was gauged by flow cytometry, measuring the expression of CD45RA and CD45RO using anti-CD45RA-APC, anti-CD45RO-PE, anti-CD3-FITC or isotype control mix (IgG2b-APC, IgG2a-PE and IgG1-FITC) antibodies (all BD Biosciences). The data were analysed on a BD FACScan using BD CellQuest software (BD Biosciences). Cultures were set up such that at each time point, a count was made and cell concentrations were re-adjusted based on the number of viable cells as described under ‘Results’ section.
7
CFSE Proliferation Assay with Cytokine Stimulation
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CFSE staining was done using 5 µM CFSE (Invitrogen, Cat# C34554) as described by Parish et al. [42] (link) and stimulated with cytokines for 6 days prior to flow cytometric analysis. Antibodies used: CD8-APC (BD, Cat# 345775), CD3-APC (BD, Cat# 555335), CD4-APC (Biolegend, Cat# 300514), CD56-APC (eBioscience, Cat#17-0569-42), CD16-PE (Biolegend, Cat#302007), HLA-DR-PE (Biolegend, Cat# 307605), NKG2D-APC (RnD Systems, Cat# FAB139A). For intracellular staining, IL-6-PE (eBioscience, Cat#12-7069) and IFN-γ-FITC (BD, Cat# 554700) were used. Isotype controls were IgG1 APC (BD, cat # 555751), rat IgG1-PE (Invitrogen, Cat # R104) and IgG1-FITC (BD, Cat #555748). PBMCs were incubated for 6 h +/− LPS (Sigma-Aldrich, L2654, 1 µg/mL) and cells were stained using the BD Cytofix/Cytoperm Kit (BD, Cat# 554714), Golgistop (BD, Cat# 554724) or Golgiplug (BD, Cat# 555029) according to the manufacturer’s protocol. Samples were run on an Accuri C6 Flow cytometer and data were analyzed using FCS Express v. 3.
8
Characterization of Wharton's Jelly Cells
9
Phenotypic profiling of MSCs and ECs
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Phenotypic characterisation was performed on culture expanded MSCs at different passages and on cultured ECs at p4 using: CD31-FITC (#MCA1738F), CD105-PE (#MCA1557PE), CD90-PE (#MCA90PE) (all from Serotec, Kidlington, UK), CD73-PE (#550257), CD146-PE (#550315) (both from BD Pharmingen, Oxford, UK), and CD271-PE (#130-091-885, Miltenyi Biotec). The isotype controls were IgG1-FITC (#550616, BD Pharmingen) and IgG1-PE (#MCA928PE, Serotec). A total of 2×10
5 cells was stained with 5 μl FITC- or PE-conjugated antibodies, and dead cells were excluded using 2 μg/ml propidium iodide (PI, #P1304MP, Invitrogen). Cells were acquired using FACScan equipped with CellQuest software version 3.1 (BD Biosciences) and the proportions of the different fractions were calculated as a percentage of total live cells.
5 cells was stained with 5 μl FITC- or PE-conjugated antibodies, and dead cells were excluded using 2 μg/ml propidium iodide (PI, #P1304MP, Invitrogen). Cells were acquired using FACScan equipped with CellQuest software version 3.1 (BD Biosciences) and the proportions of the different fractions were calculated as a percentage of total live cells.
10
Phenotypic characterization of hDPSCs
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Flow cytometric analysis was conducted to identify the phenotype of hDPSCs. The MSC phenotyping cocktail is comprised of both positive (CD29-FITC, CD44-PE, and CD90-PE-CY5, BD Bioscience, USA) and negative (CD34-PE and CD45-PE-CY5, BD Bioscience, USA) fluorochrome-conjugated monoclonal antibodies. IgG1-FITC, IgG1-PE-CY5 and IgG1-PE (BD Bioscience, USA) were used as isotype controls. hDPSCs were suspended to 4 × 105 cells/mL, incubated with different antibodies for 30 min at 4 °C, resuspended in FACS buffer, and analyzed using a MOFloTM high-performance cell sorter (Beckman Coulter, USA).
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