The T-cell suppression assay and MDSC co-cultures were performed as previously published [19, 25] . Briefly, PBMCs from IPF patients were isolated, stained with CFSE (CellTrace, C34554; ThermoFisher Scientific, Darmstadt, Germany) and stimulated with recombinant human IL-2 (100 U•mL -1 ; BD Pharmingen, San Diego, CA, USA) and OKT3 (1 µg•mL -1 ; Biolegend, San Diego, CA, USA). PBMCs were incubated with stimulation media alone or with isolated autologous MDSC. After 4 days, cells were harvested and stained with anti-CD4-PE or anti-CD8-allophycocyanin (Biolegend). CSFE fluorescence intensity of each cell type was analysed by flow cytometry. The percentage of proliferation was analysed for CD8 + and CD4 + cells cultured alone or in conjunction with MDSC. Data were analysed with FlowJo software. After 96 h of incubation, cells were harvested and stained with anti-CD4-PE (BD Pharmingen) and anti-CD8-allophycocyanin. CFSE fluorescence intensity was analysed by flow cytometry to determine proliferation of CD4 + and CD8 + T-cells. After 96 h of incubation, cells were harvested and stained with anti-CD4-PE (BD Pharmingen) and anti-CD8-allophycocyanin. CFSE fluorescence intensity was analysed by flow cytometry to determine proliferation of CD4 + and CD8 + T-cells.
Anti cd8 allophycocyanin
Manufactured by BD
Sourced in United States
Anti-CD8-allophycocyanin is a fluorochrome-conjugated antibody that binds to the CD8 surface antigen on T cells. It is used for the identification and enumeration of CD8+ T cells in flow cytometry analysis.
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Lab products found in correlation
Anti cd19 pe, by BD (5 mentions)
Anti cd4 phycoerythrin, by BD (4 mentions)
Anti cd16 apc, by BD (4 mentions)
Anti cd56 pe, by BD (3 mentions)
Anti cd56 pe antibodies, by BD (2 mentions)
Anti cd3 fluorescein isothiocyanate, by BD (2 mentions)
Anti cd4 pe, by BD (1 mentions)
Recombinant human il 2, by BD (1 mentions)
Anti cd4 pe, by BioLegend (1 mentions)
Anti cd8 allophycocyanin, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Facscanto 2 flow cytometry system, by BD (1 mentions)
Anti cd3 fitc, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Anti cd16 phycoerythrin, by BD (1 mentions)
Anti cd56 allophycocyanin, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
N antiserum to human ig kit series, by Siemens (1 mentions)
Human th1 2 cytokine kit 2, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
9 protocols using anti cd8 allophycocyanin
1
T-cell Proliferation Assay with MDSCs
2
Lymphocyte Subset Quantification by Flow Cytometry
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EDTA anticoagulated peripheral blood as flow cytometry samples was drawn from the participants before initial treatment to determine lymphocyte and subsets. All samples were tested within 6 hours of being obtained. Briefly, CD3+/CD4+/CD8+ T-cell, CD19+ B-cell, and CD16+ CD56+ NK-cell counts (cells/μL) were measured by multiple-color flow cytometry with human monoclonal anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin (PE), antiCD8-allophycocyanin (APC), anti-CD19-PE, and anti-CD56-PE antibodies (BD Multitest) according to the manufacturer’s instructions. The cells were analyzed on a BD FACS Canto II flow cytometry system (BD Biosciences) (15 (link), 19 (link)).
3
Quantifying immune cells in COVID-19
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Samples of EDTA anticoagulated peripheral blood (2 mL) were collected from patients with COVID-19 before initial treatment. All samples were tested within 6 h of being obtained. Briefly, multiple-color flow cytometry was used to measure the CD3+/CD4+/CD8+ T-cell, CD19 + B-cell, and CD16 + CD56 + NK-cell counts (cells/μL) by human monoclonal anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin (PE), anti- CD8-allophycocyanin (APC), anti-CD19-PE, anti-CD16-APC, and anti-CD56-PE antibodies (BD Multitest) according to the manufacturer’s instructions. The cells were analyzed on a BD FACS Canto II flow cytometry system (BD Biosciences).
4
Peripheral Blood Lymphocyte Subset Analysis
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The percentage of CD3+ T cells, CD3+CD8+ T cells, and CD3−CD16+CD56+ lymphocytes were detected by peripheral blood FCM (Becton Dickinson, Franklin Lakes, NJ, USA). Murine anti-human polyclonal antibodies were used, including anti-CD3-FITC, anti-CD4-phycoerythrin, anti-CD8-allophycocyanin, anti-CD16-phycoerythrin, and anti-CD56-allophycocyanin (BD Biosciences, San Diego, CA, USA). The lymphocytes were delineated by adequate forward and sidelight scatters. CellQuest software (BD Biosciences) was used to analyze the data. The peripheral blood ALC was obtained from the hematology laboratory records, and the percentages of the subtypes were determined using FCM; ACD4C refers to the CD3+CD4+ lymphocyte count, ACD8C refers to the CD3+CD8+ lymphocyte count, and ANKC refers to the CD16+CD56+ lymphocyte count in peripheral blood (15 (link)).
5
Comprehensive Immune Profile Assessment
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Cellular immunity was assessed by multicolor flow cytometric determination of surface markers using human monoclonal anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin (PE), anti-CD8-allophycocyanin (APC), anti-CD19-PE, anti-CD16-APC, and anti-CD56-PE antibodies (BD Multitest), for determination of proportions and numbers of total T (CD3+), helper T (Th, CD3+CD4+), cytotoxic T (Tc, CD3+CD8+), Natural Killer (NK, CD3-CD16+CD56+), and B (CD3-CD19+) cell subsets. All samples were examined by a BD FACSCanto II Flow Cytometer. Data were analyzed by FlowJo v10.0.
Serum levels of C reactive protein (hypersensitive, hsCRP), immunoglobulin (IgG, IgM, IgA, and IgE), and complements (C3, C4) were detected by rate nephelometry immunoassay (N Antiserum to Human Ig Kit series, Siemens, Germany). The plasma levels of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ) were detected by Cytometric Bead Array using the human Th1/2 cytokine kit II (BD Ltd., USA). All tests were conducted according to the manufacturer's instructions.
Serum levels of C reactive protein (hypersensitive, hsCRP), immunoglobulin (IgG, IgM, IgA, and IgE), and complements (C3, C4) were detected by rate nephelometry immunoassay (N Antiserum to Human Ig Kit series, Siemens, Germany). The plasma levels of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ) were detected by Cytometric Bead Array using the human Th1/2 cytokine kit II (BD Ltd., USA). All tests were conducted according to the manufacturer's instructions.
6
Chimeric Protein Vaccination Induces Immune Response
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C57BL/6 mice were i.m. vaccinated with chimeric protein (1–15 μg) alone or mixed with 400 μg of γ-PGA, 400 μg of alum, and 800 μg of PGA/alum on days 0, 14, and 28. Two weeks after the last vaccination, the mice were sacrificed, and splenocytes were obtained from the mice. Splenocytes (1 × 106 cells/100 μl) were blocked with 1 μl of anti-CD16/32 monoclonal antibody (mAb; BD Biosciences, USA) and surface-stained with an antibody mixture containing 1 μl of anti-CD3e-PerCP (BD Biosciences), anti-CD4-FITC (BD Biosciences), and anti-CD8-allophycocyanin (BD Biosciences). After incubation for 30 min at 4°C, surface-stained cells were washed twice with PBS. Then, cells were fixed and permeabilized with a BD Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s instructions. Fixed and permeabilized cells were stained with 1 μl of anti-IFN-γ-PE (BD Biosciences) for 30 min at 4°C, and cells were detected by FACSVerse or FACS Calibur flow cytometry (BD Biosciences). FlowJo software (Tree Star, USA) was used for data analysis.
7
COVID-19 Lymphocyte Subset Analysis
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Lymphocyte subset percentages were analyzed before initial treatment. EDTA anticoagulated peripheral blood (2 ml) were collected from patients with COVID-19 on admission (18 (link)). All samples were tested within 6 h of being obtained. Briefly, CD3+ total T-lymphocytes, CD19+ B-cell, CD3+/CD4+/CD8+ T-cell, and CD16+CD56+ NK-cell counts (cells/μl) were measured by multiple-color flow cytometry with human monoclonal anti-CD19-PE, anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin (PE), anti-CD8-allophycocyanin (APC), anti-CD16-APC, and anti-CD56-PE antibodies (BD Multitest) according to the manufacturer's instructions. The cells were analyzed on a Partec Cube 6 Flow cytometry system (Sysmex Europe GmbH, Germany).
8
Lymphocyte Subset Analysis in Gastric Cancer
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Peripheral blood samples (two mL) were collected from 51 GC patients and 50 healthy individuals in EDTA anticoagulant tubes. The lymphocyte subsets, including CD3+, CD4+, CD8+ T cells, CD19+ B cells, and CD16+ CD56+ NK cells, were quantified in cells/µL using a six-color flow cytometry assay. Human monoclonal antibodies, including anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin (PE), anti-CD8-allophycocyanin (APC), anti-CD19-PE, anti-CD16-APC, and anti-CD56-PE (BD Multitest), were used according to the manufacturer’s instructions. The cell samples were analyzed on a BD Canto II flow cytometry system (BD Biosciences, East Rutherford, NJ, USA).
9
Quantification of Cytokine Levels and T-Cell Subsets
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Antibodies against IL-6, p53 and IL-17R were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human recombinant IL-6 (Cat. no. 206-IL) and IL-17A (Cat. no. 7955-IL), and human neutralizing antibodies to IL-6 (aIL-6) (Cat. no. AF-206-NA) and IL-17A (aIL-17A) (Cat. no. AF-317-NA) were obtained from R&D Systems (Minneapolis, MN, USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-17A, IL-6, IL-21 and TGF-β were purchased from BioLegend (San Diego, CA, USA). Fluorescence-activated cell sorting (FACS) human antibodies, including anti-CD3-phycoerythrin (PE)-Cy7 (Cat. no. 557851), anti-CD8-allophycocyanin (Cat. no. 561952), anti-CD4-fluorescein isothiocyanate (Cat. no. 561005), anti-Foxp3-PE (Cat. no. 560852), anti-IL-17A-PE (Cat. no. 560436), anti-IL-17A-PE-Cy7 (Cat. no. 560799) and their matched anti-mouse IgG1 K-PE (Cat. no. 551436)/PE-Cy7 (Cat. no. 552811) were all purchased from BD Biosciences (San Jose, CA, USA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) reagents were obtained from Takara (Beijing, China). Rituximab was purchased from Novartis (Basel, Switzerland).
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