Isolated PBMC or cultured cells were analyzed for expression of surface markers using multi-color flow cytometry. Cell aliquots were treated with anti-human Fc mAb for 20 minutes and stained for 30 minutes with selected combinations of fluorochrome-conjugated antibodies: anti-human CD4-Alexa-PE 700 (Invitrogen, Carlsbad, CA), CD8-APC/Cy7 (BD Pharmingen, San Jose, CA), CD28-PE (BD Pharmingen, San Jose, CA), CD45RO-PerCP/Cy5.5 (BioLegend, San Diego, CA), CD25-PE/Cy7 (BioLegend, San Diego, CA), CD127-APC (BioLegend, San Diego, CA), ICOS-PerCP/Cy5.5 (BioLegend, San Diego, CA), IL15Rα (CD215)-PerCP (R&D systems, Minneapolis, MN), and CD107a-PE/Cy5 (BD Pharmingen, San Jose, CA). The cells were analyzed on a BD LSR II (BD Biosciences, San Jose, CA). Flow cytometry data was analyzed by FlowJo software version 9.4.5 (Tree Star, Inc. Ashland, OR).
Cd107a pe cy5
Manufactured by BD
Sourced in United States
CD107a-PE/Cy5 is a flow cytometry reagent that contains the CD107a (LAMP-1) antibody conjugated to the PE/Cy5 fluorochrome. CD107a is a cell surface protein that is expressed on the membrane of cytotoxic granules and is commonly used as a marker for the degranulation of these granules, which is an indicator of cell-mediated cytotoxicity.
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Lab products found in correlation
Cd56 pe cy7, by BD (3 mentions)
Golgistop, by BD (3 mentions)
Tnf α pe, by BD (2 mentions)
Cd16 apc cy7, by BD (2 mentions)
Cd3 alexa fluor 700, by BD (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Cd28 pe, by BD (1 mentions)
Cd25 pe cy7, by BioLegend (1 mentions)
Cd45ro percp cy5, by BioLegend (1 mentions)
Cd127 apc, by BioLegend (1 mentions)
Lsr 2, by BD (1 mentions)
Cd8 apc cy7, by BD (1 mentions)
Ifn γ fitc, by BD (1 mentions)
Il 2 apc, by BD (1 mentions)
Perfix nc kit, by Beckman Coulter (1 mentions)
Cd4 efluor 450, by BD (1 mentions)
Cd8 efluor 450, by BD (1 mentions)
Cxcr2 pe, by BD (1 mentions)
Cd8 pe cy7, by BD (1 mentions)
Cd4 pe cy7, by BD (1 mentions)
7 protocols using cd107a pe cy5
1
Multi-Color Flow Cytometry for Immune Cell Profiling
2
Multiparametric T cell Characterization
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T cells were washed in staining buffer (SB) consisting of phosphate-buffered saline (PBS) containing 0.1% human serum albumin (HSA) and 0.1% sodium azide before staining for 20 min at RT. The cells were then washed in SB and fixed in SB containing 1% paraformaldehyd. For intracellular staining, T cells were stimulated for 6 h or overnight with APCs, loaded or not with p573, at a T-cell to target ratio of 1:2 and in the presence of BD GolgiPlug and BD Golgistop at a 1/1,000 dilution. Cells were stained both extracellular and intracellular using the PerFix-nc kit according to the manufacturer's instructions (Beckman Coulter Inc., USA). The following antibodies were used: Vβ3- FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor450, CD4-eFluor 450, CD4-PE-Cy7, CD8-APC, CD8-eFluor 450, CD8-PE-Cy7, CD56-PE-Cy5.5 (BD Biosciences, USA) and CD107a-PE-Cy5 (BD Biosciences, USA), CXCR2-PE, IFNγ-FITC, IL-2-APC, TNF-α-PE (BD Biosciences, USA), CD261/TRAIL-R4-PE (BD Biosciences, USA). MART-1 (aa 26–35)-specific TCR was detected with dextramer staining (Immudex) following the manufacturer's recommendations. All antibodies were purchased from eBioscience, except where noted. Cells were acquired on a BD LSR II flow cytometer and the data analyzed using FlowJo software (Treestar Inc.).
3
NK Cell Degranulation Assay for SIV Antibodies
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Plates were coated with SIVmac239 gp140 and incubated overnight at 4°C. All coated wells were washed and blocked with 5% BSA in PBS for 1 h at 37°C. Plates were then washed and diluted plasma samples were added into the corresponding wells for 1 h at 37°C. Simultaneously, CD107a PE Cy5 (BD Biosciences, #555802), brefeldin A (Sigma, #B7651), and GolgiStop (BD Biosciences, #554724) were incubated with human NK cell culture for 2 h. After both incubations were completed, we added the stimulated NK cells to the plates (50,000 cells per well), and incubated them for 5 h at 37°C. Next, NK cells were transferred to V-bottom plates containing CD56 PE Cy7 (BD Biosciences, # 557747), CD16 APC Cy7 (BD Biosciences, #55775), and CD3 Alexa Fluor 700 (BD Biosciences, #557943) and incubated for 15 min at room temperature. Plates were then washed and resuspended in PBS. Samples were acquired using a LSRII (BD Biosciences). Human NK cells were isolated using a RossetteSep NK cell enrichment kit (StemCell Technologies, #15065). NK cells were defined as CD3−/CD16+ and/or CD56+. Ab-dependent NK degranulation was calculated as the percentage of NK cells positive for CD107a.
4
Phenotyping NK Cell Activation
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Enzyme-linked immunosorbent assay plates were coated with measles antigen and incubated with samples diluted 1:20, or PBS as negative control, for 2 hours at 37°C. Natural killer cells were isolated from buffy coats collected from blood bank donors. CD107a-PE/Cy5 (BD), brefeldin A (Sigma), GolgiStop (BD), and NK cells were added to the antigen-coated plate and incubated for 5 hours at 37°C. Next, cells were transferred to a V-bottom plate and stained with CD56-PE/Cy7, CD16-APC/Cy7, and CD3-AlexaFluor700 (all BD) for 15 minutes at room temperature. Cells were washed and fixed in Fixation Medium A (Life Technologies) before intracellular staining with MIP-1β-PE and IFN-γ-APC (BD) in Permeabilization Medium B (Life Technologies) for 15 minutes at room temperature. The percentages of positive NK cells for CD107a, IFN-γ, and MIP-1β were determined by flow cytometry.
5
Flow Cytometric Analysis of Cytotoxic T Cell Response
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K562 (HLA-A2) or Granta-519 cells were loaded with peptide overnight at 37 °C in a 5% CO2 incubator. Effector cells were stimulated with target cells at an effector-to-target (E:T) ratio of 1:2 for 5 hours at the same conditions as above. Conjugated CD107a was added to the cells prior to incubation. Irrelevant or no peptide served as a negative control.
The following antibodies were used: Vβ3- FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor450, CD56- eFluor, CD107a-PE-Cy5, TNFα-PE (BD Biosciences, USA), IL2-APC, IFNγ-FITC (eBiosciences, ThermoFischer). Cells were washed in flow buffer (FB, phosphate buffered saline (PBS) with 2% human bovine serum albumin (BSA) and 0.5 µM EDTA). For dextramer and antibody staining, cells were incubated for 30 minutes at room temperature (RT) with the recommended dilution in FB. If fixed, cells were incubated in FB containing 1% paraformaldehyde. For intracellular staining Perm/Wash Buffer was used (BD Biosciences) according to manufacturer’s protocol. All antibodies were purchased from eBioscience, USA, except where noted. Cells were acquired on a BD FACSCanto II flow cytometer and the data analyzed using FlowJo software (Treestar Inc., Ashland, OR, USA). Plotting and statistical analysis were performed using GraphPad prism software (La Jolla, CA, USA).
The following antibodies were used: Vβ3- FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor450, CD56- eFluor, CD107a-PE-Cy5, TNFα-PE (BD Biosciences, USA), IL2-APC, IFNγ-FITC (eBiosciences, ThermoFischer). Cells were washed in flow buffer (FB, phosphate buffered saline (PBS) with 2% human bovine serum albumin (BSA) and 0.5 µM EDTA). For dextramer and antibody staining, cells were incubated for 30 minutes at room temperature (RT) with the recommended dilution in FB. If fixed, cells were incubated in FB containing 1% paraformaldehyde. For intracellular staining Perm/Wash Buffer was used (BD Biosciences) according to manufacturer’s protocol. All antibodies were purchased from eBioscience, USA, except where noted. Cells were acquired on a BD FACSCanto II flow cytometer and the data analyzed using FlowJo software (Treestar Inc., Ashland, OR, USA). Plotting and statistical analysis were performed using GraphPad prism software (La Jolla, CA, USA).
6
Phenotypic Analysis of PBMC Subsets
7
Analyzing NK Cell Cytokine Production
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Purified NKG2D+NK cells from eight HCC patients and eight HD were sorted by flow cytometry (BD, FACSAriaII) and stimulated with PMA (25 ng/mL) (Sigma-Aldrich, P1585) and Ionomycin (1 μg/mL) (Santa Cruz Biotechnology, 56092-82-1) at 37°C for 1 h. Then, Pecy5-CD107a (BD Biosciences, 555802), GolgiStop (BD Biosciences, 554715) and Brefedlin A (Santa Cruz Biotechnology, sc-200861) were added into the medium. After 5 h, cells were collected and stained with the following monoclonal surface Abs, eFluor®450-CD3 (eBioscience, 48-0038-42), PE-CD14 (BD Biosciences, 555398), Pecy7-CD56 (BD Biosciences, 335791), APC-NKG2D (BD Biosciences, 558071). Then, cells were intracellularly stained with Alexa Fluor® 700-IFNγ (BD Biosciences, 557995) and FITC-TNF-α (BD Biosciences, 554512) and detected by flow cytometry.
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