Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml phytohaemagglutinin (Sigma) for 2 days and then rested for 1 day. Cells were pretreated with DMSO or MB or 25 μg/ml pembrolizumab or 20 μg/ml nivolumab for 1 h and then seeded in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28, with media supplemented with 10 μg/ml of hPD‐L1 protein. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with saponin (sigma, 47036). Cells were stained with CD8‐FITC (1:100, eBioscience, 11‐0081‐82), IL‐2‐PE (1:100, BioLegend, 500306), IFNγ‐PE (1:100, BioLegend, 502508), perforin‐PE (1:100, BioLegend, 353303), or GZMB‐Alexa Fluro (1:100, BioLegend, 515405), and cytokine production was measured by flow cytometric analysis by gating on CD8‐positive population.
Perforin pe
Manufactured by BioLegend
Sourced in United States
Perforin-PE is a fluorochrome-conjugated antibody that binds to the perforin protein. Perforin is a pore-forming protein found in the cytolytic granules of cytotoxic T cells and natural killer cells, and plays a crucial role in cell-mediated cytotoxicity. The Perforin-PE antibody can be used to detect and quantify perforin-expressing cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ pe, by BioLegend (2 mentions)
Ifn γ fitc, by BD (2 mentions)
Cd56 bv510, by BioLegend (2 mentions)
Brefeldin a, by BD (2 mentions)
Cd8 fitc, by Thermo Fisher Scientific (1 mentions)
Il 2 pe, by BioLegend (1 mentions)
Saponin, by Merck Group (1 mentions)
Phytohaemagglutinin, by Merck Group (1 mentions)
Gzmb alexa fluro, by BioLegend (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Fix perm buffer, by Thermo Fisher Scientific (1 mentions)
Cd107a pe cy7, by BioLegend (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Alexa fluor 647 annexin 5, by BioLegend (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
7 aad viability staining solution, by BioLegend (1 mentions)
Human nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Fixation buffer, by BioLegend (1 mentions)
Annexin 5 binding buffer, by BioLegend (1 mentions)
8 protocols using perforin pe
1
PD-1/PD-L1 blockade modulates CD8+ T-cell responses
2
RNK001 Cytotoxicity Assay
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RNK001 potency was determined by challenging them with K562 cells. Briefly, 150.000 RNK001 cells were co-cultured with 100.000 K562 cells in the presence of CD107a-PECy7 (Biolegend, #328618) at 37 °C for 4 h. After 1 h, Brefeldin A (BD Bioscience, #5550229) was added. Next, cells were harvested and stained with CD56-BV510 (Biolegend, # 318340) and eFluor780 (eBiosciences, #65-0865-14) at 4 °C for 30 min. Cells were then fixed and permeabilized using eBiosciences Fix/Perm buffer (#00-5123-43, #005223-56 and #00-8333-56). Finally, cells were stained intracellularly for IFN-γ-FITC (BD bioscience, #554700) and Perforin-PE (Biolegend, #308106), washed, acquired on the Gallios flowcytometer (Beckmann Coulter) and analyzed using Kaluza V2.1.3.
3
Comprehensive NK Cell Characterization
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Anti-human CD3-FITC/CD16 + 56-PE mixed antibody were purchased from Beckman Coulter (Brea, CA, USA). Anti-human CD16-FITC, NKG2D-PE, NKp44-APC, DNAM-1-FITC, NKp46-Alexa Fluor 647, NKp30-PE, KIR3DL1-FITC, KIR2DL1/DS1-PE, NKp30-APC, Perforin-PE and Granzyme B-FITC antibodis were all purchased from BioLegend (San Diego, CA, USA), as well as Fixation Buffer, Wash Buffer, Annexin V binding buffer, Alexa Fluor 647 Annexin V and 7-AAD Viability Staining Solution. The MPP-9 and IDO inhibitors were 1-Methyl-DL-tryptopan (1-MT; Sigma-Aldrich, St. Louis, MO, USA) and Tissue inhibitor of metalloproteinases 1 (TIMP-1; PeproTech, Rocky Hill, NJ, USA). Human NK Cell Isolation Kit was purchased from Miltenyi Biotec (Auburn, CA, USA) and ELISA kits were purchased from Abcam (Cambridge, MA, USA). Trizol reagent and PrimeScript RT Master Mix (Perfect Real Time) were both obtained from TaKaRa (Shiga, Japan) and Power SYBR Green PCR Master mix was purchased from Applied Biosystems (Carlsbad, CA, USA).
4
Flow Cytometry Antibody Panel for Immune Profiling
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The following antibodies were used for flow cytometry: purchase from Invitrogen: CD3-APC-efluor780 (145-2C11), CD4-AlexaFluor 700 (GK1.5), CD4-Pacific Orange (RM4-5), CD8-Super Bright 645 (53-6.7), NKp46-eFluor 660 or AlexaFluor 647(29A1.4), NK1.1-Super Bright 436 (PK136), GATA-3-PeCy5 (TWAJ), PD-1-SuperBright 780 (J43), Foxp3-AlexaFluor 488 (FJK-16s), CD103-AlexaFluor700 (2E7), RoRγt-PE-eFluor610 (B2D), PLZF-PeCy7 (9E12), TNF-α-PerCP-eFluor710 (MP6-XT22), IL-17-PeCy7 (eBio17B7), IL-13-Pe-eFluor610 (eBio13A), CD127-PeCy5 (A7R34), Granzyme B-eFluor450 (NGZB); purchased from Biolegend: T-bet-Brilliant Violet 605 (4B10), CD69-Brilliant Violet 711 (H1.2F3), CD44-Brilliant Violet 421 (IM7), IFN-γ-Brilliant Violet 510 (XMG1.2), TCR γδ-Pe (UC7-13D5), IL-4-Brilliant Violet 711 (11B11), IL-22-APC (Poly5164), ki67-PerCP/Cy5.5 (16A8), CXCR3-Brilliant Violet 510 (CXCR3-173), CXCR6-Brilliant Violet 421 (SA051D1), Perforin-Pe (S16009B), B220-Brilliant Violet 750 (RA3-6B2), F4/80-Brilliant Violet 785 (BM8), Ly6G/Ly6C-Alexa Fluor 700 (RB6-8C5). Appropriate isotype controls were also purchased.
The α-GalCer-CD1d-tetramer-AlexaFluor 488, the MHC class I NS41602-1611 tetramer-APC and the MHC class I NS31043-1052 tetramer PE were obtained from the NIH tetramer core facility.
The α-GalCer-CD1d-tetramer-AlexaFluor 488, the MHC class I NS41602-1611 tetramer-APC and the MHC class I NS31043-1052 tetramer PE were obtained from the NIH tetramer core facility.
5
Tumor Tissue Dissociation and Immune Cell Analysis
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To obtain single-cell suspensions, the mouse tumor tissues were minced and digested, and then filtered through 70μm strainers. Erythrocytes in the cell suspension were lysed by RBC lysis buffer (Biolegend, USA), and the supernatant was removed by centrifugation. Cells were washed with PBS and stained for flow cytometry with the following antibodies: CD3-APC, CD45-PE-cy7/FITC, CD4-FITC, CD8-PE/pecy7, CD11b-PE, GR1-PC5.5, F4/80-APC, CD206-FITC, His-APC, Ly6G-PC5.5, TREM2-PE, PD-1-FITC, IFN-γ-PE, PE Goat anti-mouse IgG, Granzyme B-PE, and Perforin-PE (BioLegend).
6
NK Cell Cytokine and Cytotoxic Profiling
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For IFNγ content, HPC-NK cells were stimulated for 4 h with K562, THP-1 or SKOV-3 at an E:T ratio of 1.5:1, in the absence or presence of 1 nM N-803, 1 nM rhIL-15, or 1000 U/ml rhIL-2 (Chiron, NDC 53905–991-01) and in the presence of brefeldin A (added after 1 h, BD Biosciences, 555029). For perforin production, PB-NK cells and HPC-NK cells were primed overnight with or without 1 nM N-803.
After stimulation, surface staining was performed of CD56-BV510 (Biolegend, 318340), and intracellular staining of perforin-PE (Biolegend, 308106) and IFNγ-FITC (BD Biosciences, 554700). Dead cells were excluded using Fixable Viability Dye eFluor780. IFNγ analysis was performed by gating on CD56+ perforin+ NK cells, using unstimulated cells as control. Perforin analysis was performed by gating on CD56+ NK cells.
After stimulation, surface staining was performed of CD56-BV510 (Biolegend, 318340), and intracellular staining of perforin-PE (Biolegend, 308106) and IFNγ-FITC (BD Biosciences, 554700). Dead cells were excluded using Fixable Viability Dye eFluor780. IFNγ analysis was performed by gating on CD56+ perforin+ NK cells, using unstimulated cells as control. Perforin analysis was performed by gating on CD56+ NK cells.
7
Comprehensive Immune Cell Profiling
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Peripheral blood mononuclear cells were incubated with directly conjugated antibodies for 30 min at 4°C. The cells were then washed before flow cytometric analysis. Antibodies used were anti‐human CD3‐BV786, CD4‐APC‐H7, CD8‐BV510 or CD8‐BV421, CD45RA‐AF700, CCR7‐BV421, CD95‐PE‐CF594, PD‐1‐BV711, BTLA‐BV650, TIM‐3‐BV650, CD160‐AF488, 2B4‐FITC, CD107a BV421 (BD Biosciences, San Diego, CA, USA), CD226‐FITC, Granzyme B‐AF700, perforin‐PE (BioLegend, San Diego, CA, USA), TIGIT‐PE‐Cy7, and LAG‐3‐APC (Ebioscience, San Diego, CA, USA) and the corresponding isotype controls. Data acquisition was performed on an LSR Fortessa flow cytometer (BD Biosciences), and data analysis was performed using FlowJo Software (Tree Star, Ashland, OR, USA).
8
Characterizing Murine T-cell Phenotypes
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