Expanded CD8+ T cells were stimulated for 3–4 hours with MART-126–35 A27L peptide-loaded T2 cells or melanoma cell lines. Golgi plug (protein transport inhibitor containing brefeldin A, BD) and Golgi stop (Monensin, BD) were added 2 hours before staining. Cells were stained for cell surface markers followed by intracellular cytokines (IFNγ [4S. B3, BD], TNFα [MAb11, BD], and IL-2 [MQ1–17H12, BD]) after fixation and permeabilization. For perforin (B-D48, BioLegend) and granzyme B (GB11, BD) staining, effector cells were cocultured with T2 cell for 16 hours. To analyze cytokine production of T cells, effector cells were cocultured with T2 cells for 24 hours and supernatants were collected. Cytokines were analyzed using a Luminex system (ThermoFisher Scientific).
Mq1 17h12
Manufactured by BD
The MQ1-17H12 is a laboratory equipment product manufactured by BD. It is designed to perform specific functions within a laboratory setting. The core function of this product is to facilitate laboratory processes, but a detailed description cannot be provided while maintaining an unbiased and factual approach without further information.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ 4s b3, by BD (2 mentions)
Mab11, by BD (2 mentions)
Fixation permeabilization solution kit, by BD (2 mentions)
Golgistop monensin, by BD (1 mentions)
Granzyme b gb11, by BD (1 mentions)
Perforin b d48, by BioLegend (1 mentions)
Brefeldin a, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Mh9a4, by BioLegend (1 mentions)
Uchl1, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Annexin 5, by BD (1 mentions)
Lsrii 5 laser flow cytometer, by BD (1 mentions)
Anti cd4 apc alexafluor750, by Beckman Coulter (1 mentions)
Anti cd8 percp cy5, by BD (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Anti cd28 cd28, by Thermo Fisher Scientific (1 mentions)
Fab210f, by R&D Systems (1 mentions)
Specific antibody, by BD (1 mentions)
Cd62l clone dreg56, by BD (1 mentions)
7 protocols using mq1 17h12
1
Characterizing MART-1 Specific CD8+ T Cells
2
Comprehensive T cell Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunophenotype of T cells was performed using standard staining and flow cytometry techniques as described before41 (link),42 (link). Briefly, combinations of fluorophore-conjugated anti-human monoclonal antibodies specific for CD3 (BioLegend, OKT3, Cat#317344, 1:500), CD4 (BioLegend, OKT4, Cat#317438, 1:500), CD8 (BioLegend, SK1, Cat#344724, 1:500), CCR7 (BioLegend, G043H7, Cat#353214, 1:500), CD45RO (BioLegend, UCHL1, Cat#304244, 1:500), PD1 (eBioscience, EBIOJ105, Cat#12-2799-42, 1:500), TIM3 (BioLegend, F38-2E2, Cat#345006, 1:500), LAG3 (eBioscience, 3DS223H, Cat#15-2239-42, 1:500) were used to label T cells in flow cytometry staining buffer for 30 min on ice after Fc blocking, followed by intracellular cytokine staining with the BD Fixation/Permeabilization Solution Kit. Data were acquired on LSRFortessa (BD Biosciences) and analyzed with FlowJo software (Treestar). Cytokine antibodies included IFN-γ (eBioscience, B27, Cat#MHCIFG04, 1:500), IL9 (BioLegend, MH9A4, Cat#507614, 1:500), IL2 (BioLegend, MQ1-17H12, Cat#500342, 1:500), GrzB (BD Biosciences, GB11, Cat#561142, 1:500), or annexin V (BD Bioscience, Cat#550475, 1:50).
3
Cytokine Responses in Infant Immunity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole blood cytokine levels and intracellular cytokine staining were conducted on heparinized blood drawn from 10-week old South African infants. 10 milliliters of blood were taken and used both for the cytokine assays and genotyping. Samples were stimulated ex-vivo with media or BCG (strain SSI, 1.2 x 106 CFU’s/ml) for 7 hours at 37°C. For whole-blood assays, IL-2 and IFN- γ were measured by multiplex bead array technology according to manufacturer’s instructions (Bio-Rad, Hercules, CA, USA) and read on a Luminex luminometer (Luminex, Austin, TX, USA). Basal cytokine levels measured in plasma harvested from unstimulated blood were subtracted from values obtained from BCG-stimulated blood [11 (link)].
For intracellular cytokine staining, Brefeldin A was added to the above samples and cells were incubated for 5 additional hours. Samples were frozen in 40% RPMI and 40% DMSO with 10% FCS and thawed prior to the time of data analysis. Flow cytometry was performed at the University of Washington Flow Cytometry Core Facility on an LSRII 5-laser flow cytometer (Becton Dickenson, Inc) using antibodies against cell surface markers including: anti-CD3 ECD (Beckman Coulter, UCHT1), anti-CD4 APC-Alexa Fluor 750 (Beckman Coulter, 13B8.2), and anti-CD8 PerCP Cy5.5 (BD, SK1). Antibodies against cytokines included anti-IL-2 PE (BD, MQ1-17H12) and anti-IFN-γ (BD, B27) [33 (link)].
For intracellular cytokine staining, Brefeldin A was added to the above samples and cells were incubated for 5 additional hours. Samples were frozen in 40% RPMI and 40% DMSO with 10% FCS and thawed prior to the time of data analysis. Flow cytometry was performed at the University of Washington Flow Cytometry Core Facility on an LSRII 5-laser flow cytometer (Becton Dickenson, Inc) using antibodies against cell surface markers including: anti-CD3 ECD (Beckman Coulter, UCHT1), anti-CD4 APC-Alexa Fluor 750 (Beckman Coulter, 13B8.2), and anti-CD8 PerCP Cy5.5 (BD, SK1). Antibodies against cytokines included anti-IL-2 PE (BD, MQ1-17H12) and anti-IFN-γ (BD, B27) [33 (link)].
4
Cytokine Production in CD3+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD3+ T cells were treated for 72 hours with CRP at the indicated concentrations, in the presence of IL-2 (100 IU/mL), then stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA) and ionomycin (plus protein transport inhibitors) (Invitrogen). Cells were stained for cell surface markers followed by intracellular cytokines (interferon (IFN)-γ (4S.B3, BD Biosciences), tumor necrosis factor (TNF)-α (MAb11, BD Biosciences) and IL-2 (MQ1-17H12, BD Biosciences)) after fixation and permeabilization.
5
Multiparametric Flow Cytometry of T Cell Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used: Alexa Fluor 700–conjugated CD4 (RPA-TA; BD), allophycocyanin (APC)-Cy7–conjugated CD25 (M-A251; BD), PE-Cy5–conjugated CD127 (eBioRDR5; eBioscience), Pacific blue–conjugated CD14 (M5E2; BD), FITC-conjugated Ki67 (35 Ki-67; BD), PE-conjugated RORγτ (AFKJS-9; eBioscience), Alexa Fluor 647–conjugated FoxP3 (PCH101; eBioscience), PE-conjugated FoxP3 (236A/E; eBioscience), FITC-conjugated membrane TNF (FAB210F; R&D Systems), PE-conjugated TNF-RII (22235; R&D Systems), PE-conjugated STAT5 (PY694; BD), FITC-conjugated active Caspase 3 (C92-605; BD), FITC-conjugated IL-2 (MQ1-17H12; eBioscience), Alexa Fluor 647–conjugated Il-17A (BL168; BioLegend), PE-Cy7–conjugated IFN-γ (B27; BD), soluble anti-CD3 (HIT-3a; eBioscience), anti-CD28 (CD28.2; eBioscience), anti–TNF-RII antagonist (22221; R&D Systems), anti–TNF-RII agonist (MR2-1; Hycult Biotech), and anti–IL-2 neutralizing mAb (MQ1-17H12; BD). STAT5 inhibitor N′([4-oxo-4H-chromen-3-yl]methylene) nicotinohydrazide (EMD Millipore) was also used. Of relevance, FITC-conjugated membrane TNF (FAB210F; R&D Systems) does not interfere with membrane TNF’s binding to TNF-RII (Gerspach et al., 2000 (link)).
6
Multicolor flow cytometry analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T cells were tested for the expression of CD3 (clone SK7), CD8 (clone RPA-T8), CD4 (clone SK3), CD56 (clone B159), CD62L (clone DREG-56) and CD45RO (clone UCHL1) using specific antibodies (BD Bioscience, San Jose, CA), whereas leukemic blasts were assessed for CD45 (clone 2D1), CD33 (clone HIM3-4), CD123 (clone 7G3), CD19 (clone HIB19) expression (BD Bioscience) and CD10 (eBioCB-CALLA, eBioscience, San Diego, CA). For intracytoplasmic staining, T cells were stained with anti-CD3 mAb before fixation, permeabilization (Fixation/Permeabilization Solution Kit, BD Bioscience) and incubation with anti-human IFN-γ (B27) and IL-2 mAbs (MQ1-17H12, BD Pharmingen, San Diego, CA). CAR expression was detected using an anti-Human IgG (H+L) specific antibody (Jackson ImmunoResearch, Suffolk, UK), as previously described. [49 (link)] Samples were acquired using a BD FACS Canto flow cytometer (BD Biosciences), and data were analyzed with FlowJo 7.5.5 (Tree Star, Inc., Ashland, OR) and BD FACSDIVA™ (BD Biosciences). Quadrant markers were set accordingly to unstained controls.
7
SARS-CoV-2 Spike Protein T-cell Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryopreserved peripheral blood mononuclear cells (PBMC) were thawed and rested overnight. PBMC (1 × 10 6 per well) were stimulated with a SARS-CoV-2 spike overlapping peptide pool (JPT, 1 μg/mL each peptide; 0.4% final DMSO concentration), 0.4% DMSO as a negative control, or PHA-P (Remel, Lenexa, KS, USA; 1.6 μg/mL final) as a positive control, in the presence of anti-CD28 (1:1000, L293, BD Biosciences) and anti-CD49d (1:1000, L25, BD Biosciences) antibodies at 37°C for 6 hours. Brefeldin A (Sigma) was added after 2 hours. Cells were stained with Live/Dead Near IR dye (1:250, Invitrogen), treated with FACS lyse (BD Biosciences) and frozen at -80°C. For staining, the cells were thawed, washed, and permeabilized with Permeabilizing solution 2 (BD Biosciences) then stained with fluorochrome labeled monoclonal antibodies to anti-human CD3-PE-Texas Red (1:40, UCHT, Beckman Coulter), anti-human CD4-BV510 (1:83, SK3, Biolegend), anti-human CD8-PerCP-Cy5.5 (1:33, SK1, BD Biosciences), and the activation markers anti-human CD40L-BV421 (1:167, TRAP1, BD Biosciences), anti-human IFNγ-PE (1:500, 4S.B3, BD Biosciences), anti-human IL2-APC (1:250, MQ1-17H12, BD Biosciences), and anti-human TNFα-FITC (1:400, Mab11, BD Biosciences). Events were recorded with BD Fortessa and analyzed with FlowJo (v10 for Mac; BD).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!