Antibody-dependent NK cell degranulation was conducted as previously described59 (link). NVX-CoV2373 Spike protein was coated on Maxisorp ELISA plate (Thermo Fisher), and then blocked with 5% BSA. Antibodies were then added and incubated for 2 hours at 37°C. Human NK cells were isolated from peripheral blood by negative selection using the RosetteSep Human NK cell enrichment cocktail following the manufacturer’s instructions. Human NK cells were then added to the bound antibody and incubated for 5 hours at 37°C in the presence of RMPI+10%FBS, GolgiStop (BD), Brefeldin A (Sigma), and anti-human CD107a antibody (BD Bioscience). After incubation, cells were washed, stained with CD16, CD56, and CD3 (BD Bioscience), and fixed in 4% PFA for 15 minutes. Intracellular staining was performed using the FIX/PERM Cell fixation and permeabilization kit (Thermo), and cells were stained for interferon-γ and macrophage inflammatory protein-1β (BD bioscience). Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1).
Anti human cd107a antibody
Manufactured by BD
The Anti-human CD107a antibody is a laboratory reagent used for the detection and analysis of CD107a, a protein expressed on the surface of cells. This antibody is commonly used in flow cytometry and other immunoassays to identify and characterize CD107a-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Forecyt, by Intellicyt (3 mentions)
Interferon γ and macrophage inflammatory protein 1β, by BD (3 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (3 mentions)
Fix perm cell fixation and permeabilization kit, by Thermo Fisher Scientific (3 mentions)
Brefeldin a, by Merck Group (3 mentions)
Golgistop, by BD (3 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Golgistop, by Merck Group (1 mentions)
Recombinant human ifnα, by BioLegend (1 mentions)
Recombinant human il 12, by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Golgiplug protein transport inhibitor, by BD (1 mentions)
Dxflex flow cytometry, by Beckman Coulter (1 mentions)
Kaluza analysis 2, by Beckman Coulter (1 mentions)
Affinipure f ab 2 fragment specific goat anti mouse igg antibody, by Jackson ImmunoResearch (1 mentions)
Streptavidin pe, by BD (1 mentions)
Il 12, by Thermo Fisher Scientific (1 mentions)
Quanti luc gold substrate, by InvivoGen (1 mentions)
Jurkat lucia nfat, by InvivoGen (1 mentions)
7 protocols using anti human cd107a antibody
1
Antibody-Dependent NK Cell Degranulation Assay
2
Antibody-dependent NK Cell Degranulation
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Antibody-dependent NK cell degranulation was conducted as previously described.83 (link) NVX-CoV2373 Spike protein was coated on Maxisorp ELISA plate (Thermo Fisher), and then blocked with 5% BSA. Diluted serum (1:25 dilution) was then added and incubated for 2 h at 37°C. Human NK cells were isolated from peripheral blood by negative selection using the RosetteSep Human NK cell enrichment cocktail following the manufacturer’s instructions. Human NK cells were then added to the bound antibody and incubated for 5 h at 37°C in the presence of RPMI+10%FBS, GolgiStop (BD), Brefeldin A (Sigma), and anti-human CD107a antibody (BD Bioscience). After incubation, cells were washed, stained with CD16, CD56, and CD3 (BD Bioscience), and fixed in 4% PFA for 15 min. Intracellular staining was performed using the FIX/PERM Cell fixation and permeabilization kit (Thermo), and cells were stained for interferon-γ and macrophage inflammatory protein-1β (BD bioscience). Flow cytometry was performed with an iQue (IntelliCyt) and analysis was performed on IntelliCyt ForeCyt (v8.1) (Figure S1 ).
3
Stimulating PBMC Cytokine Responses
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Freshly isolated PBMCs from healthy individuals were cultured for 16 h in RPMI 1640 medium containing 20% foetal bovine serum (Sigma) in the presence of the following stimuli: 1000 U/mL recombinant human IFN-α (Biolegend) or 10 ng/mL recombinant human IL-12 (Peprotech) plus 100 ng/mL IL-15 (Peprotech). GolgiStop (Sigma) was added to the medium for 4 h for stimulation, and antihuman CD107a antibody (BD) was added for 2 h for stimulation. Cells were cultured in a medium alone as a negative control.
4
Characterization of Gene-edited CAR T Cells
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Gene‐edited Jurkat CAR19 and CAR T‐19 cells were incubated with a biotin‐SP‐AffiniPure F(ab)’2 fragment‐specific goat anti‐mouse IgG antibody (Jackson ImmunoResearch) to assess CAR expression, followed by incubation with streptavidin‐PE (BD Biosciences). In addition, the gene‐edited Jurkat CAR19 and CAR T‐19 cells were incubated with anti‐human B2M, CD3 (BD Biosciences), HLA‐ABC, HLA‐C, HLA‐E, and HLA‐G (Biolegend) antibodies.
Gene‐edited CAR T‐19 cells were washed and stained with anti‐human CD3, CD4, CD8, CD45RO, and CD62L antibodies (BD Biosciences).
Gene‐edited CAR T‐19 cells were cocultured with Raji or K562 cells at an effector‐to‐target (E:T) ratio of 1:1 together with an anti‐human CD107a antibody (BD Biosciences) for 1 h to assess degranulation, followed by incubation with a Golgi Plug protein transport inhibitor (BD Biosciences) for 3 h.
Primary NK cells were incubated with anti‐human CD3, CD56 (BD Biosciences), NKG2A, KIR2DL4, and LILRB1 (Biolegend) antibodies.
All of the antibody information is presented in Supporting Information Table1 . All samples were assessed using DxFLEX flow cytometry (Beckman Coulter). The data were analyzed with Kaluza Analysis 2.0 (Beckman Coulter) and FlowJo software version 10 (FlowJo LLC).
Gene‐edited CAR T‐19 cells were washed and stained with anti‐human CD3, CD4, CD8, CD45RO, and CD62L antibodies (BD Biosciences).
Gene‐edited CAR T‐19 cells were cocultured with Raji or K562 cells at an effector‐to‐target (E:T) ratio of 1:1 together with an anti‐human CD107a antibody (BD Biosciences) for 1 h to assess degranulation, followed by incubation with a Golgi Plug protein transport inhibitor (BD Biosciences) for 3 h.
Primary NK cells were incubated with anti‐human CD3, CD56 (BD Biosciences), NKG2A, KIR2DL4, and LILRB1 (Biolegend) antibodies.
All of the antibody information is presented in Supporting Information Table
5
PBMC Stimulation Assay
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Thawed PBMC were plated at 1 million cells by well in a 96-well U-bottom plate. PBMC were left unstimulated or incubated in the presence of IL-12 (10 ng/mL, Peprotech) and IL-18 (100 ng/mL, MBL) for 24 h in R10 media. Anti-human CD107a antibody (BD Biosciences) was added for the entire duration of the culture. Monensin and brefeldin A were added for the last 6 h of culture before staining of the samples.
6
Antibody-Mediated NK Cell Cytotoxicity Assay
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Antibody-dependent NK cell degranulation was conducted as previously described59 (link). NVX-CoV2373 Spike protein was coated on Maxisorp ELISA plate (Thermo Fisher), and then blocked with 5% BSA. Antibodies were then added and incubated for 2 hours at 37°C. Human NK cells were isolated from peripheral blood by negative selection using the RosetteSep Human NK cell enrichment cocktail following the manufacturer’s instructions. Human NK cells were then added to the bound antibody and incubated for 5 hours at 37°C in the presence of RMPI + 10%FBS, GolgiStop (BD), Brefeldin A (Sigma), and anti-human CD107a antibody (BD Bioscience). After incubation, cells were washed, stained with CD16, CD56, and CD3 (BD Bioscience), and fixed in 4% PFA for 15 minutes. Intracellular staining was performed using the FIX/PERM Cell fixation and permeabilization kit (Thermo), and cells were stained for interferon-γ and macrophage inflammatory protein-1β (BD bioscience). Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1).
7
Jurkat CAR-T Cell Activation and Degranulation
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Reporter cell line Jurkat-NFAT-Lucia (Invivogen, France) expressing CD19-CAR (Jurkat CD19-CAR) or IL13-CAR (Jurkat IL13-CAR) were obtained by lentiviral transduction as described before. For activation assay 5x10 4 Jurkat CAR-T cells were mixed with freshly isolated antigen-positive or antigen-negative AVs at CAR-T cell-to-AVs cell ratios (0, 4:1, 2:1, 1:1, 1:2, 1:5, 1:10 or 1:20) in a 96-well plate and incubated for 24 hours at 37°C. 25µl of supernatant was separated from cells by centrifugation (4°C, 300g, 5 min) and transferred to the opaque 96-well plate. Activation of reporter Jurkat-NFAT-Lucia CAR-T cells was measured by level of luciferase activity following reaction with Quanti-luc Gold substrate (Invivogen, France) according to the manufacturer's instructions.
T cell degranulation assay: IL13-CAR T cells were resuspended in TexMACS media in concentration 2x10 6 cells/ml. 100 µl of cell suspension was transferred to the 96-well plate and 2µl of anti-human CD107a antibody (BD) were added in each well. CAR-T cells were mixed with either antigen-positive or antigen-negative AVs at ratio 1:1, or kept untreated (ctl) and were incubated for 2 hours on 4°C. Cell suspension was washed twice with TexMACS media (4°C, 300g, 5 min), stained with anti-human CD3/CD4/CD8 antibodies and analyzed by flow cytometry.
T cell degranulation assay: IL13-CAR T cells were resuspended in TexMACS media in concentration 2x10 6 cells/ml. 100 µl of cell suspension was transferred to the 96-well plate and 2µl of anti-human CD107a antibody (BD) were added in each well. CAR-T cells were mixed with either antigen-positive or antigen-negative AVs at ratio 1:1, or kept untreated (ctl) and were incubated for 2 hours on 4°C. Cell suspension was washed twice with TexMACS media (4°C, 300g, 5 min), stained with anti-human CD3/CD4/CD8 antibodies and analyzed by flow cytometry.
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