The characterization of PBMCs that were co-cultured for 1 hour with Vero E6 and TZM-bl cell monolayers was performed by staining with the following conjugated antibodies purchased from BD Biosciences (San Jose, CA): CD3-PE, CD8-APC-H7, CD56-BV605, TCRγδ-FITC, and CD107a-PE-Cy7. CD107a was used as a degranulation marker and its expression at the cell surface peaks within 1 hour of target cell engagement (35 (link)), being afterwards actively recycled from the cell surface (36 (link)). CD3+CD8- were assumed to be CD4+ T cells, which included those cells with downregulated CD4 expression caused by HIV-1 infection (37 (link)). Isotype controls were used to determine the background signal. Data acquisition was performed with BD LSRFortessa X-20 flow cytometer (BD Biosciences) and data analysis with FlowJo software v10.0.7 (Tree Star Inc.). Gating strategy is shown in Supplementary Figure 1
Cd107a pe cy7
Manufactured by BD
Sourced in United States
CD107a-PE-Cy7 is a fluorescently-labeled antibody used to detect and quantify the expression of the CD107a (LAMP-1) surface antigen on cells. CD107a is a lysosome-associated membrane protein that is transiently expressed on the cell surface during degranulation and is commonly used as a marker for cell activation and cytotoxicity.
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Lab products found in correlation
Lsrfortessa x 20 flow cytometer, by BD (6 mentions)
Cd8 apc h7, by BD (6 mentions)
Cd3 pe, by BD (4 mentions)
Cd16 percp, by BD (4 mentions)
Cd56 bv605, by BD (3 mentions)
Tcrγδ fitc, by BD (3 mentions)
Cd56 fitc, by BD (3 mentions)
Polybrene, by Merck Group (2 mentions)
Facsdiva software, by BD (2 mentions)
Brefeldin a, by BD (2 mentions)
Cd3 apc, by BD (2 mentions)
Cd4 percp, by BD (2 mentions)
Cd25 pe cy5, by R&D Systems (2 mentions)
Cd127 fitc, by R&D Systems (2 mentions)
Cd4 percp, by R&D Systems (2 mentions)
Cd28 cd49d antibodies, by BD (1 mentions)
Pkh67 green fluorescent cell linker, by Merck Group (1 mentions)
Facsdiva software v6, by BD (1 mentions)
Cd3 bv510, by BD (1 mentions)
Pd 1 bv650, by BD (1 mentions)
11 protocols using cd107a pe cy7
1
Characterization of PBMC Interactions
2
HIV Infection Dynamics in Stimulated CD4 T Cells
3
CD4 T cell HIV infection and CTL response
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HLA-B57+ primary CD4 T cells were either treated with LRA, stimulated with anti-CD3/CD28 or kept in culture with no stimulation for 48 hours. The cells were thoroughly washed and magnetic anti-CD3/CD28 beads were removed. For each condition, 2 million of CD4 T cells were infected with 20 ug HIV Gag p24 equivalent of NL4-3-Δenv-GFP virus pseudotyped with Vesicular Stomatitis Virus glycoprotein (VSV-g) in the presence of 5ug/mL polybrene (Sigma) by spinfection for 1.5 h at 2000x g as in [37 (link)]. At 24, 48 and 72 h post-infection, CD4 T cells were plated with epitope-specific CD8 T cells at a ratio of 1:4 (CD4:CD8) and an aliquot of CD4 T cells was also harvested to measure HIV-1 infection through GFP and HIV-1 p24 intracellular expression, as well as cellular activation and HLA class I surface expression by flow cytometry. After 30 min of co-culture, CD107a-Pe-Cy7 (BD Biosciences) and CD28/CD49d antibodies (BD Biosciences) were added to each well to measure CTL degranulation. After 6 h cells were harvested for surface staining.
The avidity of the CTL clones was assessed by performing a concentration-course titration. Target HLA-B57 CD4 T cells were incubated with increasing concentration of the cognate peptides (0.000002–2 ug/mL) before co-culture with the corresponding CD8 T cells as described above.
The avidity of the CTL clones was assessed by performing a concentration-course titration. Target HLA-B57 CD4 T cells were incubated with increasing concentration of the cognate peptides (0.000002–2 ug/mL) before co-culture with the corresponding CD8 T cells as described above.
4
Measuring ADCC Capacity of Patient PBMCs
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The Raji cell line was used as target to measure ADCC capacity of PBMCs of patients and donors, as described before [39 (link)]. Briefly, Raji cells were previously labeled with PKH67 Green Fluorescent Cell Linker (Merck KGaA, Darmstadt, Germany) and then coated with rituximab (50 μg/mL) (Selleckhem, Houston, TX, USA) for 4 h. Labeled Raji cells were then cocultured for 18 h with PBMCs (1:2 ratio) from the recruited patients and healthy donors. Apoptosis of Raji cells was determined by staining with Annexin V conjugated with phycoerythrin (PE) (Immunostep, Salamanca, Spain). Cytotoxic cell populations such as natural killer (NK), NKT-like and TCRγδ+ cells were analyzed in the supernatants using specific conjugated antibodies: CD3-PE, CD56-BV605, CD16-PercP, CD8-APC H7, CD107a-PE-Cy7, and TCRγδ-FITC (BD Biosciences). Data acquisition was performed in a BD LSRFortessa X-20 flow cytometer and FACS Diva software (BD Biosciences). FlowJo software (Tree Star Inc.) was used for data analysis.
5
Multiparameter Flow Cytometry of Cell Subsets
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For staining of cell surface phenotyping markers, the following conjugated antibodies were used: CD3-PE, CD8-APC-H7, CD56-FITC, and CD107a-PE-Cy7, purchased from BD Biosciences (San Jose, CA, USA). For staining of exhaustion markers, the following conjugated antibodies were used: CD3-BV510, CD8-APC-H7, CD56-FITC, and PD1-BV650, purchased from BD Biosciences, as well as CD4-PE, purchased from Immunostep S.L. (Salamanca, Spain) and TIGIT-AlexaFluor700 purchased from Thermo Fisher (Waltham, MA, USA). Samples were acquired by using BD LSRFortessa X-20 flow cytometer with FACS Diva software v 6.0 (BD Biosciences, Franklin Lakes, NJ, USA) and then analyzed using FlowJo software v10.0.7 (Tree Star Inc., Ashland, OR, USA).
6
Multiparametric Flow Cytometry Assay
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Mixed cell suspensions were activated with phorbol 12-myristate 13-acetate (PMA) (100 ng/ml, Abcam) and ionomycin (2 μM, Calbiochem) for 1 h in the presence of CD107a-PE-Cy7 (BD Bioscience) antibody, followed by 4 additional hours in the presence of Brefeldin A (BD GolgiPlug protein transport inhibitor, BD Biosciences) as described before (20 (link)), surface stained and fixed and permeabilized with the BD Cytofix/Cytoperm kit (BD Biosciences) according to the instructions. Intracellular staining of perforin, GZA and GZB was performed as described below.
7
Multi-Parametric Immunophenotyping of T-Cell Subsets
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For staining of cell surface markers, the following conjugated antibodies were used: CD3-APC (BD Biosciences; San Jose, CA), CD4-PercP (BD), CD8-APC-H7 (BD), CD16-PercP (BD), CD56-FITC (BD), CD107a-PE-Cy7 (BD), TCRγδ-PE (BioLegend, London) and CD158f/KIR2DL5-BV421 (R&D Systems) for analysis of CD4 + conventional T-cells (CD4 + T), CD8 + conventional T-cells (CD8 + T), NK, and NKT cell populations. CD4-PercP, CD25-PE-Cy5 and CD127-FITC (R&D Systems) for CD4 + natural regulatory T-cells (Treg) and CCR7-FITC (Biosciences) and CD45RA-PE-Cy7 (Biosciences) for CD4 + , CD8 + T cell memory subpopulations that were determined as follows: naïve (CD45RA+CCR7 +), central memory (TCM) (CD45RA-CCR7 +), effector memory (TEM) (CD45RA-CCR7-) and terminally differentiated effector memory (TEMRA) (CD45RA+CCR7-) cells. Samples were acquired on BD LSRFortessa X-20 flow cytometer (BD Biosciences, San Jose, CA) and analyzed using Flow Jo software v10.0.7 (Tree Star Inc., Ashland, OR, USA).
8
Intracellular Cytokine Staining Workflow
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Intracellular cytokine staining was performed as previously described(1) using the following markers: CD3-APCH7, CD107a PECy7, CD14-Pacific Blue, CD16-Pacific Blue, CD4-PECy5.5, IFN-γ-PerCPCy5.5, CD45RO-AF700, CD19-Pacific Blue (BD Biosciences, San Jose, CA), TNFα-AF647, CD8-BV570, CCR7-BV711 (BioLegend, San Diego, CA), granzyme B-PE Texas Red (Invitrogen), CD27-PECy5 (eBioscience, San Diego, CA) perforin-FITC(Abcam, Cambridge, UK). Prepared cells were acquired using an LSR II flow cytometer equipped with BD FACSDiva software (BD Biosciences). Acquired data was analyzed using the FlowJo software version 7.6.3 (Tree Star).
9
Multiparametric Flow Cytometry Analysis of Immune Cells
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Conjugated antibodies for surface staining CD3-APC, CD4-PercP, CD8-APC-H7, CD16-PercP, CD56-FITC, CD107a-PE-Cy7, NKp44-BUV395, and NKp46-BUV650 were purchased from BD Biosciences (San Jose, CA), whereas PD1-BUV650, NKG2A-PE, and NKG2C-Alexa Fluor700 were purchased from R&D Systems (Minneapolis, MN). Tregs cells were characterized by staining with CD4-PercP, CD25-PE-Cy5 and CD127-FITC (R&D Systems). CD4+ and CD8+ T cell subpopulations were determined by staining with CCR7-FITC and CD45RA-PE-Cy7 (Biosciences) as follows: naïve (CD45RA+CCR7+), central memory (TCM) (CD45RA-CCR7+), effector memory (TEM) (CD45RA-CCR7-) and terminally differentiated effector memory (TEMRA) (CD45RA+CCR7-) cells.
For intracellular staining of granzyme B (GZB) from NK and NKT cells, PBMCs were treated for 4 h at 37°C with Hsp70 peptide 1 µgr/ml (Abcam, Cambridge, UK) to stimulate cytolytic activity of NK cells, in the presence of brefeldin A (BD Biosciences). Cells were then stained with antibodies against CD3, CD56 and CD16 conjugated to APC, FITC and PercP, respectively. After fixation and permeabilization with IntraPrep Permeabilization Reagent (Beckman Coulter), cells were stained with an antibody against GZB-PE (BD Biosciences) and then acquired and analyzed in a BD LSRFortessa X-20 flow cytometer (BD Biosciences) using FACS Diva (BD Biosciences) and FlowJo_V10 software (TreeStar).
For intracellular staining of granzyme B (GZB) from NK and NKT cells, PBMCs were treated for 4 h at 37°C with Hsp70 peptide 1 µgr/ml (Abcam, Cambridge, UK) to stimulate cytolytic activity of NK cells, in the presence of brefeldin A (BD Biosciences). Cells were then stained with antibodies against CD3, CD56 and CD16 conjugated to APC, FITC and PercP, respectively. After fixation and permeabilization with IntraPrep Permeabilization Reagent (Beckman Coulter), cells were stained with an antibody against GZB-PE (BD Biosciences) and then acquired and analyzed in a BD LSRFortessa X-20 flow cytometer (BD Biosciences) using FACS Diva (BD Biosciences) and FlowJo_V10 software (TreeStar).
10
NK Cell Functional Assay in CLL
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PBMC samples from both CLL patients and HC were enriched for NK cells by CD19 depletion, the percentage of NK cells was determined by flow cytometry, and cells were rested O/N in an incubator at 37°C. The next day, cells were co-cultured with K562 or Daudi cells (NK:target ratio of 1:10) or stimulated with PMA/Ionomycin, or left untreated for 4 hours at 37°C in the presence of brefeldin A (10 μg/ml, Sigma, St Louis, MO), GolgiStop and CD107a PE-Cy7 (BD Biosciences). Afterwards, cells were washed with PBA, and stained with CD56 BUV395, CD3 V500 (BD Biosciences) and Live/Dead Fixable Red Stain (Invitrogen) for 30 minutes at 4°C. Then cells were washed with PBA, fixed and permeabilized (Cytofix/Cytoperm reagent, BD Biosciences) and stained intracellularly for 30 minutes at 4°C with IFNγ BV421, pS6 (Ser240/244, Cell Signaling, Danvers, MA) and granzyme B AF700 (BD Biosciences). Cell were washed, resuspended in PBA, and analyzed on a LSR Fortessa flow cytometer (BD Biosciences). Data analysis was performed using Flowjo Mac Version 10. Gating strategy can be found in supplemental Figure 5B (Supplemental Digital Content).
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