ADNKA assay was performed as described with modifications (Gunn et al., 2020 (link)). ELISA plates were coated with recombinant RBD antigen (300 ng/well) (Bates et al., 2021c ) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) in duplicate for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 104 cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences). Cells were stained with anti-CD56 (clone 5.1H11, BD Biosciences) and anti-CD16 (clone 3G8, BD Biosciences) and fixed with 4% PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BD Biosciences) and TNFα (clone Mab11, BD Biosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed in all cells. NK cell degranulation and activation were calculated as percent of CD56+NK cells positive for CD107a, or IFNγ or TNFα expression. Representative data from one dilution was chosen by the highest signal to noise ratio for further analyses.
Clone mab11
Manufactured by BD
Clone Mab11 is a laboratory equipment product designed for specific applications. It serves a core function within the intended workflow, but a detailed description while maintaining an unbiased and factual approach is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Clone b27, by BD (3 mentions)
Permeabilization buffer, by BioLegend (2 mentions)
Anti cd107a clone h4a3, by BD (2 mentions)
Anti cd56 clone 5.1h11, by BD (2 mentions)
Anti cd16 clone 3g8, by BD (2 mentions)
Brefeldin a, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Clone 4s b3, by BD (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Pta 6967, by American Type Culture Collection (1 mentions)
Interleukin 2 (il 2), by Roche (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Golgiplug, by BD (1 mentions)
Il 15, by Miltenyi Biotec (1 mentions)
Ccr7 pe cy7, by BD (1 mentions)
Cd45ra pe, by BD (1 mentions)
Ifn γ fitc, by BD (1 mentions)
Clone hil 7r m21, by BD (1 mentions)
Clone mq1 17h12, by BD (1 mentions)
5 protocols using clone mab11
1
NK Cell-Mediated ADNKA Assay for Antigen-Specific Responses
2
ADNKA Assay for SARS-CoV-2 RBD Antibodies
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ADNKA assay was performed as described with modifications (Gunn et al., 2020 (link)). ELISA plates were coated with recombinant RBD antigen (300 ng/well) (Bates et al., 2021c (link)) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 104 cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences). Cells were stained with anti-CD56 (clone 5.1H11, BD Biosciences) and anti-CD16 (clone 3G8, BD Biosciences) and fixed with 4% PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BD Biosciences) and TNFα (clone Mab11, BD Biosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed in all cells. NK cell degranulation and activation were calculated as percent of CD56+NK cells positive for CD107a, or IFNγ or TNFα expression. Representative data from one dilution was chosen by the highest signal to noise ratio for further analyses.
3
Stimulation and Analysis of Tumor-Infiltrating Lymphocytes
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Tumor-infiltrating lymphocytes were stimulated for 5 h with plate-bound anti-CD3 (Miltenyi Biotec MACS GMP, clone OKT3) and soluble anti-CD28 (Miltenyi Biotec, clone 15E8) antibodies in the presence of GolgiPlug (BD Biosciences) in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum. After stimulation, intracellular cytokine staining was performed to assess IFNy (BD Biosciences, clone B27) and TNFα (BD Biosciences, clone Mab11). For CD226 downregulation and induction assays, cells were stimulated for 6 days with TGF-β (50 ng/ml, Peprotech), IL15 (50 ng/ml, Miltenyi Biotec) or IL2 (20 UI/ml, Roche). For STAT inhibitor experiments, cells were incubated for 48 h with IL15 in the presence of STAT3i (CAS 501919-59-1, Sigma‒Aldrich) or STAT5i (CAS 2062-78-4, Sigma‒Aldrich).
4
Comprehensive Immune Cell Profiling
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The following conjugated antibodies were used for cell-surface staining: CD3-Pacific Blue (BD Pharmingen; Clone UCHT1; Cat. No. 558117; San Diego, CA, USA), CD8-APC H7 (BD Pharmingen; Clone SK1; Cat. No. 641400), CD45RA-PE (BD Pharmingen; Clone HI100; Cat. No. 555489), CCR7-PE-Cy7 (BD Pharmingen; Clone 3D12; Cat. No. 557648), CD28-PerCP-Cy5.5 (BD Biosciences; Clone L293; Cat. No. 337181; San Jose, CA, USA), CD27-Alexa Fluor 700 (BD Pharmingen; Clone M-T271; Cat. No. 560611), CD95-APC (BD Pharmingen; Clone DX2; Cat. No. 558814) and CD127-FITC (BD Pharmingen; Clone HIL-7R-M21; Cat. No. 560549). Conjugated antibodies for intracellular staining included the following: IFN-γ-FITC (BD Pharmingen; Clone 4S.B3; Cat. No. 554551), IL-2-PerCP-Cy5.5 (BD Pharmingen; Clone MQ1-17H12; Cat. No. 560708) and TNF-α-AlexaFluor 700 (BD Pharmingen; Clone MAb11; Cat. No. 557996). To exclude dead cells, the Fixable Aqua Dead Cell Stain viability marker was used (Invitrogen; Cat. No. L34957; Eugene, OR, USA).
5
Multicolor Flow Cytometry Analysis of Immune Cell Subsets
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PBMCs were washed with FACS buffer and stained for cell viability with the fixable live/dead Aqua staining kit (Molecular Probes) in the presence of Fc-R blocking reagent (Miltenyi biotech), and the antibodies against the surface markers anti-CD3-APC, CD45-V450 and anti-CD11b-APC/Cy7 indicated above. Surface staining was carried out for 25 min at 4°C. Cells were washed, and intracellular staining was carried out with BD cytofix-cytoperm kit (BD Biosciences). Cells were fixed with Fix-Perm (BD Biosciences) for 20 min at room temperature and permeabilized with Perm-Wash (BD Biosciences) for 30 min at room temperature. Cells were stained with two combinations of antibodies diluted in Perm-Wash: anti-IFN-γ (PE, 1:100, clone 4S.B3, BD Biosciences), anti-IL-4 (FITC, 1:100, clone MP4-25D2, BD Biosciences), and anti-IL-17A (PE/Cy7, 1:100, clone BL168, BioLegend) or anti-TNF-α (AlexaFluor-488, 1:100, clone MAb11, BD Biosciences) and anti-IL-10 (PE, 1:30, clone JES3-9D7, BD Biosciences). Data were acquired on a FACSCanto II flow cytometer (BD Biosciences) with Diva software (BD Biosciences) and analyzed with FlowJo (v10, BD Biosciences).
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