We cultured 2,000–5,000 MAIT cells from MAIT cells expanded in RPMI 1640 with 10% FBS or 2% Phx with 10,000–25,000 THP-1 cells to serve as APCs in RPMI with 10% FBS without cytokines overnight. We then stimulated the coculture of MAIT cells and THP-1 with 10 MOIs of strain 1100–2 fixed E. coli for 20 h and blocked extracellular transport with brefeldin A for a total of 4 h. For extracellular staining, we used Zombie ultraviolet fixable viability dye (BioLegend), anti-CD3-BUV395 (BD Biosciences), anti-CD8-BV605 (BioLegend), anti-CD4-BUV496 (BD Biosciences), anti-Vα7.2-PE-Cy7 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD69-BUV563 (BD Biosciences), anti-CD161-PE-Dazzle-594 (BioLegend), anti-TIM-3-BV421 (BioLegend), anti-CD25-BV650 (BD Biosciences), and anti-human PE-MR1–5-OP-RU tetramer (NIH Tetramer Core Facility). The cells were fixed and permeabilized using Foxp3/transcription factor kit (eBioscience) and stained intracellularly with anti-granzyme-B Alexa Fluor 700 (BioLegend), anti-TNF-α eFluor 450 (Invitrogen), and anti-IFN-γ-FITC (BioLegend). Sample data were acquired with a five-laser Cytek Aurora flow cytometer (Cytek) and analyzed using FlowJo software version 10 (BD Biosciences).
Anti cd4 buv496
Manufactured by BD
Anti-CD4-BUV496 is a fluorescently labeled antibody that binds to the CD4 cell surface protein. It can be used for the identification and enumeration of CD4-positive cells in flow cytometric applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 buv395, by BD (3 mentions)
Anti cd8 bv605, by BioLegend (3 mentions)
Anti cd161 pe dazzle 594, by BioLegend (2 mentions)
Anti granzyme b alexa fluor 700, by BioLegend (2 mentions)
Anti tnf α efluor 450, by Thermo Fisher Scientific (2 mentions)
Aurora flow cytometer, by Cytek (2 mentions)
Foxp3 transcription factor kit, by Thermo Fisher Scientific (2 mentions)
Anti vα7.2 pe cy7, by BioLegend (2 mentions)
Anti lag 3 bv786, by BioLegend (2 mentions)
Zombie ultraviolet fixable viability dye, by BioLegend (2 mentions)
Anti cd69 buv563, by BD (2 mentions)
Anti tim 3 bv421, by BioLegend (2 mentions)
Anti cd25 bv650, by BD (2 mentions)
Anti ifn γ fitc, by BioLegend (2 mentions)
Anti cd8 buv805, by BD (2 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Anti cd56 pe, by BioLegend (1 mentions)
Anti cd11b apc cy7, by BioLegend (1 mentions)
5 protocols using anti cd4 buv496
1
MAIT Cell Activation and Cytokine Production
2
MAIT Cell Activation and Cytokine Production
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We cultured 2,000–5,000 MAIT cells from MAIT cells expanded in RPMI 1640 with 10% FBS or 2% Phx with 10,000–25,000 THP-1 cells to serve as APCs in RPMI with 10% FBS without cytokines overnight. We then stimulated the coculture of MAIT cells and THP-1 with 10 MOIs of strain 1100–2 fixed E. coli for 20 h and blocked extracellular transport with brefeldin A for a total of 4 h. For extracellular staining, we used Zombie ultraviolet fixable viability dye (BioLegend), anti-CD3-BUV395 (BD Biosciences), anti-CD8-BV605 (BioLegend), anti-CD4-BUV496 (BD Biosciences), anti-Vα7.2-PE-Cy7 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD69-BUV563 (BD Biosciences), anti-CD161-PE-Dazzle-594 (BioLegend), anti-TIM-3-BV421 (BioLegend), anti-CD25-BV650 (BD Biosciences), and anti-human PE-MR1–5-OP-RU tetramer (NIH Tetramer Core Facility). The cells were fixed and permeabilized using Foxp3/transcription factor kit (eBioscience) and stained intracellularly with anti-granzyme-B Alexa Fluor 700 (BioLegend), anti-TNF-α eFluor 450 (Invitrogen), and anti-IFN-γ-FITC (BioLegend). Sample data were acquired with a five-laser Cytek Aurora flow cytometer (Cytek) and analyzed using FlowJo software version 10 (BD Biosciences).
3
TIL-Tumor Cell Co-culture Assay
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Fresh or defrosted REP TILs were counted and pre-stained in fluorescence-activated cell sorter (FACS) buffer with anti-CD4 BUV496 and anti-CD8 BUV805 (BD, Vianen, the Netherlands) for 30 min at 4°C. Cells were washed once with FACS buffer, and once with 20/80 T-cell mixed media. A total of 1 × 105 live expanded TILs were co-cultured with 2 × 105 live tumor digest cells for 6 h at 37°C. Alternatively, expanded TILs were stimulated with 10 ng/ml phorbol myristate acetate (PMA) and 1 μg/ml ionomycin (Sigma-Aldrich), or cultured with T cell mixed media alone. After 1 h of co-culture, 1x Brefeldin A and 1x Monensin (Invitrogen, Landsmeer, the Netherlands) were added.
4
Postmortem Leptomeningeal Cell Analysis
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Fresh postmortem leptomeninges from an ALS case were enzymatically dissociated as described above and stained with anti-CD11b:APC-Cy7 (Biolegend cat. #101226), anti-CD3:BUV395 (BD Biosciences cat. #563546), anti-CD4:BUV496 (BD Biosciences cat. #612936), anti-CD8:BV605 (Biolegend cat. #301040), anti-CD45RO:BV421 (Biolegend cat. #304224), anti-CD45RA:BV711 (Biolegend cat. #304138), anti-CD69:PerCP (Biolegend cat. #310927), anti-CD103:BV785 (Biolegend cat. #350229), anti-CXCR6:FITC (Biolegend cat. #356019), and anti-CD56:PE (Biolegend cat. #318306), antibodies in 2% FBS in PBS for 20 minutes. Stained samples were subsequently spun at 300g for 10 minutes and resuspended in 2% FBS containing PI at a dilution of 1:1000. Data were immediately acquired on the Novocyte Penteon.
5
Comprehensive Immunophenotyping of PBMC Subsets
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Frozen PBMCs from human healthy donors were processed as for the HS1 datasset and stained with live/dead marker and fluorochrome-conjugated antibodies against the following surface markers: anti-CD8-BUV805, anti-CD4-BUV496, anti-CD95-BUV737, anti-CD28-BB660-P, anti-ICOS-BB630, anti-CXCR3-BV785, anti-PD-1-BV750-P, anti-CXCR5-BV650, anti-CCR2-BV605, (all BD Biosciences); anti-CD3-PerCP-Vio700 (Mil-tenyi Biotec); anti-CD45RA-FITC, anti-CD14-PE-Cy5.5, fixable viability dye eFluor780 (all eBioscience); anti-CD25-BV711, anti-CD31-BV480, anti-HLA-DR-BV570, anti-CD127-BV421, anti-CCR4-PE/Dazzle 594, anti-CCR7-PE-Cy7 (all BioLegend).
Samples were stained for 60 min at 4
Samples were stained for 60 min at 4
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