All experimental groups were infected or not with 103 LM-OVA for 7 days. At 7 days post-infection, spleens were harvested, processed to a single-cell suspension, and stained individually with anti-mouse CD8 antibody (BD Biosciences, 563898) and H2-Kb-SIINFEKL Dextramer (Immudex, Copenhagen, Denmark, JD2163) according to manufacturer’s instruction. Samples were analyzed by FACS using BD FACSCelestaTM (BD, Mountain View, CA, United States). Each sample was analyzed independently by using the gating strategy shown in Supplementary Figure S1 . Finally, the frequency of CD8+ H2-Kb–SIINFEKL+ population of each sample was evaluated separately using FlowJo v10 workspace.
H2 kb siinfekl dextramer
Manufactured by Immudex
The H2-Kb-SIINFEKL Dextramer is a lab equipment product used in flow cytometry applications. It is a multimeric MHC-peptide complex designed for the detection and analysis of antigen-specific T cells.
Automatically generated - may contain errors
Lab products found in correlation
Lympholyte m, by Cedarlane (2 mentions)
Facscelesta, by BD (1 mentions)
Anti mouse cd8 antibody, by BD (1 mentions)
Facscanto 2, by Tree Star (1 mentions)
Cell strainer, by BD (1 mentions)
Tumor digestion kit, by Miltenyi Biotec (1 mentions)
Cd8 bv510, by BioLegend (1 mentions)
Cd4 bv421, by BioLegend (1 mentions)
Anti cd45 buv395, by BD (1 mentions)
Cd44 percp cy5, by BioLegend (1 mentions)
Cd62l af700, by BioLegend (1 mentions)
Brilliant stain buffer, by BD (1 mentions)
Zombie nir, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
5 protocols using h2 kb siinfekl dextramer
1
Quantifying CD8+ T Cell Response
2
Multiparametric Flow Cytometry Analysis
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Antimouse antibodies for CD45 (30-F11), CD4 (GK1.5 or RM4-5), CD8a (53–6.7), CD44 (IM7), CD127 (SB/199), CD62-L (MEL-14), KLRG1 (2F1), IFNγ (XMG1.2), TNFα (MP6-XT22), IL-2 (JES6-5H4), CD107a (1D4B), FoxP3 (FJK-16s) were obtained from eBioscience (Paris, France) or BD Biosciences (San Jose, CA). H2Kb-SIINFEKL Dextramer, a multimer of H2Kb loaded with OVA257-264 antigen was purchased at Immudex (Copenhagen, Denmark). Staining was performed as described by manufacturer’s instruction. The Fixable Viability Dye (FVD eF780, eBioscience) was used for tumor-infiltrated study in order to exclude dead cells for flow cytometry analysis. Flow cytometry acquisition was done using a BD Biosciences FACSCanto II and analysis carried out with FlowJo software (Tree Star, Ashland, OR).
3
Evaluating Combination Immunotherapy in Mice
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Mice were implanted with MC38OVA-GFP cells and injected intraperitoneally with SAR439459 or isotype control at 25 mpk, mouse PD-1 antibody, or isotype (MOPC-21) at 5 mpk or their combination; mice were given a total of 6 treatment doses in 3 weeks (2/week). Tumors, spleen, and tumor-draining lymph nodes were harvested, and used for preparing single-cell suspensions. Tumors were digested by tumor digestion kit as instructed (Miltenyi Biotec, 130–096-730), whereas lymph nodes and splenocytes were passed through cell strainers (Falcon, 352350). Cells were stained with the H2Kb-SIINFEKL dextramer (Immudex, JD2163-PE and JD2163-APC) as instructed by the manufacturer, followed by Fc blocking (BioLegend, 101320). An antibody cocktail was prepared by mixing anti- CD45-BUV395 (BD Bioscience, 564279), CD8-BV510 (Biolegend, 100752), CD4-BV421, (Biolegend, 100438), CD44-PerCP/Cy5.5 (Biolegend, 103032), CD62L-AF700 (Biolegend, 104426), and CD69-BV711 (Biolegend, 104537) in Brilliant Stain Buffer (BD Bioscience, 563794), and cell suspensions were stained for 30 minutes at 4°C followed by staining with dead cell exclusion dye (Zombie NIR; BioLegend, 423106) and fixed with 1% formaldehyde (Electron Microscopy Systems, 15710) diluted in PBS (GIBCO, 10010–023). Samples were acquired on LSRFortessa (BD Biosciences) and analyzed using FlowJo software.
4
Isolation and In Vitro Activation of T Cells
5
Isolation and Functional Assay of Tumor-Infiltrating T Cells
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Tumors were excised and subjected to manual homogenization. T cells were isolated by density gradient separation with Lympholyte-m (Cedarlane). For in vitro function, isolated T cells were (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Pre-existing intratumoral CD8 T cells substantially contribute to control tumors following therapeutic anti-CD40 and polyI:C based vaccination 8 stimulated with plate-bound αCD3 (1µg/mL; eBioscience) or OVA257 peptide pulsed, CD45mismatched splenocytes. H-2K b /SIINFEKL dextramer (Immudex) was used to identify OVA257 specific CD8. Antibodies and reagents used for staining are listed in table S1.
Pre-existing intratumoral CD8 T cells substantially contribute to control tumors following therapeutic anti-CD40 and polyI:C based vaccination 8 stimulated with plate-bound αCD3 (1µg/mL; eBioscience) or OVA257 peptide pulsed, CD45mismatched splenocytes. H-2K b /SIINFEKL dextramer (Immudex) was used to identify OVA257 specific CD8. Antibodies and reagents used for staining are listed in table S1.
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