The HLA-null K562 human chronic myelogenous leukemia cell line was purchased from ATCC (CCL-234) and cultured in cRPMI. Media was added every two days to maintain cell concentrations at 0.25×106 − 1.0×106 cells/mL. Cryopreserved PBMCs were thawed and pre-treated with IL-12 (1 ng/mL) and/or IL-18 (10 ng/mL) for 24 hours. K562 cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) according to the manufacturer’s instructions and then, co-cultured in a 96-well round bottom plate with 10:1, 5:1, and 2.5:1 PBMC effector to K562 target ratios. After 2.5 hours of incubation at 37°C and 5% CO2, cells were stained with Viability Mastermix. For effector phenotyping, cells were stained for surface [CD8-BV605 (RPA-T8), NKG2D-BV711 (1D11), and PD-1-AF647 (EH12.2H7) (all Biolegend)] and intracellular markers [Perforin-Pacific Blue (dG9; Biolegend), IFN-γ-PE-Cy7 (B27; BD Biosciences), Granzyme B-PE (GB11; BD Biosciences)] as described in section 2.2 . Samples were collected on a BD Accuri C6 cytometer and analyzed using FlowJo software. Percent specific lysis was calculated by subtracting background lysis (Annexin V+PI+) in wells containing 0:1 PBMC to K562 cells from lysis in wells containing effector cells.
Granzyme b pe gb11
Manufactured by BD
Sourced in United States
Granzyme B-PE (GB11) is a laboratory reagent used in flow cytometry applications. It is a fluorescently labeled antibody that detects the presence of granzyme B, a serine protease involved in cellular cytotoxicity.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 bv605 rpa t8, by BioLegend (1 mentions)
Ifn γ pe cy7, by BD (1 mentions)
Cell analysis software, by FlowJo (1 mentions)
Facsaria 3, by BD (1 mentions)
Attune nxt, by BD (1 mentions)
Cd3 pe cy7, by BD (1 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cd3 percp sk7, by BD (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Cd56 fitc, by BD (1 mentions)
Annexin 5 pe 7 aad apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using granzyme b pe gb11
1
PBMC Cytotoxicity Assay against K562 Cells
2
Comprehensive Immune Cell Phenotyping
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Antibodies for flow cytometry and cell sorting were from Biolegend unless otherwise indicated. Antibodies used were: CD161 APC (HP-3G10), CD3 PE/Cy7 (UCHT1), CD3 APC/Cy7 (HIT3a), CD38 CF594 (HIT2, BD Biosciences), CD4 Alexa Fluor® 700 (RPA-T4), CD69 Pacific Blue™ (FN50), TCR γ/δ BV421™ (B1), Granzyme B PE (GB11, BD Biosciences), IFN-γ BV786 (4S.B3, BD Biosciences), TCR Vα7.2 BV605™ or PerCP/Cy5.5 (3C10), TNF BV650 (MAb11, BD Biosciences). Dead cells were excluded using LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Life Technologies). Flow cytometry data was collected on BD LSRFortessa™ X20 or Invitrogen Attune NxT. Cell sorting was performed on BD FACSAria™ Fusion Cell Sorter or BD FACSAria™ III. Data was analyzed using FlowJo™ cell analysis software (FlowJo, LLC).
3
Phenotyping Naïve and Induced NK Cells
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To determine the purity and the surface markers of naïve and induced NK cells, the cells were adjusted to 1 × 106 cells/mL and stained with CD3-PerCP (SK7, cat # 347344), CD56-PE (B159, cat # 555516) or CD56-FITC (B159, cat # 562794), CD57-FITC (HNK-1, cat # 340706), NKG2D-APC (1D11, cat # 558071), KIR3DL1-APC (DX9, cat # 564103), KIR2DL2/3-PE (CH-L, cat # 559785), and KIR2DL1-PE (HP-3E4, cat # 556063) (all from BD Biosciences, San Jose, CA, USA) and NKG2A-PE (S19004C, cat # 375104; BioLegend) for 15 min at 23°C in the dark. The cells were washed twice with PBS and detected using a BD FACS Aria II flow cytometer (BD Biosciences) or ACEA NovoCyte D3000 flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
CD16-FITC (NKP15, cat # 335035), interferon (IFN)-γ-PE (4S.B3, cat # 554552), granzyme B-PE (GB11, cat # 561142), perforin-PE (δG9, cat # 556437), and CD107a (H4A3, cat # 641581) assays (all from BD Biosciences) were carried out as previously described (27 (link), 28 (link)). Apoptosis was measured using the Annexin V-PE/7-AAD Apoptosis Detection Kit (eBioscience, San Diego, CA, USA), as per the manufacturer’s instructions. To analyze cell-cycle progression, induced NK cells were collected and labeled with propidium iodide.
CD16-FITC (NKP15, cat # 335035), interferon (IFN)-γ-PE (4S.B3, cat # 554552), granzyme B-PE (GB11, cat # 561142), perforin-PE (δG9, cat # 556437), and CD107a (H4A3, cat # 641581) assays (all from BD Biosciences) were carried out as previously described (27 (link), 28 (link)). Apoptosis was measured using the Annexin V-PE/7-AAD Apoptosis Detection Kit (eBioscience, San Diego, CA, USA), as per the manufacturer’s instructions. To analyze cell-cycle progression, induced NK cells were collected and labeled with propidium iodide.
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