CD19 CAR transduced T cells (1×106 cells) were incubated at an effector target ratio of 1:1 with either CD19+ K562, NGFR+ K562 or autologous B cells (CLL cells or EBV-transformed B cells), in AIM-V medium supplemented with 5% heat-inactivated human AB serum in presence of 10 μg/mL brefeldin A (Sigma-Aldrich), GolgiStop (BD Biosciences) and the anti-CD107a (BD Biosciences) antibody for 6 hours at 37°C and 5% CO2. After incubation, monoclonal antibodies directed against CD3 (SK7), CD4 (RPA-T4), and CD8 (SK1) (all from BD Biosciences) were used for cell surface staining, and after fixation and permeabilization with the BD Cytofix/Cytoperm kit (BD Biosciences), antibodies against IL-2 (MQ1–17H12) (Biolegend), TNF (Mab11) (eBioscience), IFN-γ (4S.B3), and IL-17 (N49–653) (BD Biosciences), or in other experiments granzyme B (GB11) (BD Biosciences) were used for intracellular staining. For maximal stimulation controls, PMA and ionomycin (Sigma-Aldrich) were used at 25 ng/mL and 1 μg/mL, respectively, in presence of brefeldin A at 10 μg/mL and GolgiStop for 6 hours at 37°C and 5% CO2. Cells were analyzed by flow cytometry as described above.
Mq1 17h12
Manufactured by BioLegend
The MQ1-17H12 is a laboratory instrument designed for performing various scientific analysis and research tasks. It is a versatile piece of equipment that can be utilized in a wide range of applications. However, without additional details about the specific features and capabilities of this product, a more detailed and unbiased description cannot be provided while maintaining objectivity. The core function of this equipment is to support scientific investigations, but the exact nature of its applications would require further information.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (3 mentions)
Il 17, by BD (1 mentions)
Golgistop, by Merck Group (1 mentions)
Granzyme b gb11, by BD (1 mentions)
Anti cd107a, by BD (1 mentions)
Tnf mab11, by Thermo Fisher Scientific (1 mentions)
Rpa t4, by BD (1 mentions)
Ifn γ 4s b3, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Attune nxt acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
Anti cd8 clone rpa t8, by BioLegend (1 mentions)
Cd19 hib19, by BioLegend (1 mentions)
Brefeldin a, by BD (1 mentions)
Mq2 13a5, by Thermo Fisher Scientific (1 mentions)
Bl168, by BioLegend (1 mentions)
Hla dr l243, by BioLegend (1 mentions)
6 protocols using mq1 17h12
1
T Cell Cytokine Production Assay
2
Cytokine profiling of activated T cells
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1x106 cryopreserved PBMC were thawed, washed, and then stimulated for 16 hours at 37C in the presence or absence of anti-CD3/CD28 dynabeads per manufacturer’s instructions (ThermoFisher Sceintific); Brefeldin A (Sigma Aldrich, St. Louis, MO) was added after 1 incubation. Cells were stained for surface markers CD4 (OKT4, Biolegend) and CD8b (2ST8.5H7, Beckman Coulter), fixed, permeabilized, and then stained intracellularly for IFN-γ (4S.B3, Biolegend), TNF-α (Mab11, ThermoFischer Scientific), MIP-β (D21-1351, BD Pharmingen), IL-2 (MQ1-17H12, Biolegend) and IL-17 (eBio64DEC17, ThermoFisher Scientific). Samples were then acquired on the Attune NxT acoustic focusing cytometer (Life Technologies). Data were analyzed using FlowJo v10 (TreeStar, Ashland, OR USA).
3
Evaluating CAR-T Cell Cytokine Production
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CAR-T cells 15E7CH2-CH3 (2×105) and LFTT1CH2-CH3 were seeded with 2×105 K562-HLA-G1 in a U-bottom 96-well plate and centrifuged at 100 g during 1 min. Cells were incubated 1 hour at 37°C in a 5% CO2 incubator then brefeldin A (5 µg/mL, BD Biosciences) was added. Cell were incubated 18 hours longer at 37°C in a 5% CO2 incubator. For FACS analysis, cells were labeled with anti-CD8 (clone RPA-T8, Biolegend), CD19 (HIB-19, Biolegend), interferon (IFN)γ (clone 4S.B3, Biolegend), tumor necrosis factor (TNF)⍺ (clone MAb11, Biolegend), IL-2 (MQ1-17H12, Biolegend) and a viability dye.
4
Cytokine Production in T cells, Monocytes, and DCs
5
Multiparametric Flow Cytometry Assays
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To assess surface expression of CD11a and CD3ε on Jurkat cells, 0.25 × 106 cells/sample were washed and blocked with 2% FBS/PBS for 30 min at 4°C and then stained with anti-CD3ε-Alexa Fluor 488 (BioLegend, #317310) and anti-CD11a (ThermoFisher, #MA11A10) conjugated in house with Alexa Fluor 647 (ThermoFisher, #A20006), at 10 μg/mL for 30 min at 4°C. Finally, cells were washed three times in 2% FBS/PBS, fixed in 2% PFA/PBS, analyzed by LSRFortesa (BD Biosciences) with BD FACSDiva software and plotted using FlowJo version 10.
Flow cytometric analysis of surface CD69 was carried out using FITC anti-CD69, clone FN50 (BioLegend, #310904) at 1 μg/ml for 30 min at 4°C. Samples were analyzed with Guava Easy Cyte Cytometer (Millipore) and plotted using FlowJo version 6.1.1.
IL-2 intracellular staining flow cytometry was carried out using APC-labeled anti-human IL-2, clone MQ1-17H12 (BioLegend, #500310), at 0.125 μg/5 × 105 cells. Samples were analyzed using a Becton Dickinson FACS CANTO II with BD FACSDiva 6.0 software.
For all experiments unstained cells and isotype controls were performed for background correction and gating.
Flow cytometric analysis of surface CD69 was carried out using FITC anti-CD69, clone FN50 (BioLegend, #310904) at 1 μg/ml for 30 min at 4°C. Samples were analyzed with Guava Easy Cyte Cytometer (Millipore) and plotted using FlowJo version 6.1.1.
IL-2 intracellular staining flow cytometry was carried out using APC-labeled anti-human IL-2, clone MQ1-17H12 (BioLegend, #500310), at 0.125 μg/5 × 105 cells. Samples were analyzed using a Becton Dickinson FACS CANTO II with BD FACSDiva 6.0 software.
For all experiments unstained cells and isotype controls were performed for background correction and gating.
6
Cytokine Release Assay for Immunotherapies
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Cytokine release was assessed 48 hours after incubation of target cells with BiMAb, TNFL fusion proteins and purified T cells as described above. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Anti-human IFN-γ (MD-1, BioLegend), anti-human IL-2 (MQ1-17H12, BioLegend) capture antibodies were coated on a 96-well Nunc MaxiSorp™ (ThermoFisher) flat-bottom plates and incubated overnight at 4°C protected from light. After washing, blocking was performed to reduce unspecific binding. Supernatant of the cytotoxicity assay was added and incubated for 90 minutes at room temperature. For detection, biotinylated anti-human IFN-γ (4S.B3, BioLegend) or anti-human IL-2 (Poly5176, BioLegend) antibodies were used in combination with streptavidin-horseradish peroxidase (HRP; BioLegend) and TMB (3,3’,5,5’-tetramethyl benzidine) substrate solution. The reaction was stopped with H2SO4 and the absorbance was measured at 450 and 540 nm. Cytokine concentrations were calculated based on reference cytokine standards (BioLegend).
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