Monocytes were purified from freshly isolated PBMCs using the EasySepTM human monocyte enrichment kit (StemCell technologies, Cat# 19059). Monocyte-enriched cells were resuspended in R10 medium and 3 x 105 cells were seeded in 96-well plates and stimulated with 20 µg/ml of cGAMP (Invivogen France). IFN-α and IP-10 concentartions were measured by ELISA in 24 hours supernatants. Alternatively, negatively-enriched monocytes were stimulated for 18 hours with cGAMP 20 µg/ml in R10 complete medium, in the presence of brefeldin A for the final 3 hours. The cells were then harvested, surface stained with CD11c-BV421 (BD Biosciences Cat# 562561, RRID:AB_2737656); HLA-DR-APC-Vio770 (Miltenyi Cat# 130-113-399, RRID:AB_2733092); fixed, permeabilized (BD Biosciences Cat# 554722, RRID:AB_2869010 and Cat# 554723, RRID:AB_2869011, respectively), and intracellularly stained with anti-IFN-a-PE (Miltenyi Cat# 130-092-601, RRID:AB_871560).
Hla dr apc vio770
Manufactured by Miltenyi Biotec
The HLA-DR-APC-Vio770 is a monoclonal antibody conjugated to a fluorescent dye. It is designed for the detection and analysis of human leukocytes expressing the HLA-DR antigen using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Easysep human monocyte enrichment kit, by STEMCELL (1 mentions)
Cd11c bv421, by BD (1 mentions)
Cgamp, by InvivoGen (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Lsr fortessa instrument, by BD (1 mentions)
Cd44 fitc, by BD (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Cd24 bv421, by BD (1 mentions)
Kaluza analysis software, by Beckman Coulter (1 mentions)
Perm wash solution, by BD (1 mentions)
Viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
Fortessa, by BD (1 mentions)
Fixation permeabilization solution, by BD (1 mentions)
3 protocols using hla dr apc vio770
1
Monocyte Activation by cGAMP Stimulation
2
Multiparameter Flow Cytometry Sorting
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Cells were detached using TrypLE Select (Thermo Fisher scientific), pelleted, and re‐suspended in FACS buffer (0.5% BSA/2 mM EDTA in PBS), followed by staining with fluorophore‐conjugated antibodies for 20 min, 4°C. Antibodies used were CD24‐BV421 (BD PharMingen, #562789), CD44‐FITC (BD PharMingen, #555478), and HLA‐DR‐APC‐Vio770 (Miltenyi Biotec, #130‐111‐792). Isotype control antibodies for each fluorophore were from the same companies as the primary antibodies. Cell sorting was performed on a BD FACSAria III Cell Sorter and flow cytometry on a BD LSR Fortessa instrument (BD Biosciences, for instrument settings, see Appendix Supplementary methods ). Data analysis was performed using the Kaluza Analysis Software (Beckman Coulter). The optical configuration on the BD FACSAriaIII cell sorter and the BD LSR Fortessa flow cytometer is presented in Table S2 .
3
Multi-parameter immune cell profiling
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Cells were stained for viability with Viability Dye-eFluor506 (eBiosciences). For human's surface markers, cells were stained in FACS solution with the following antibodies: BDCA4-PB (clone 12C2, Biolegend), and HLA-DR-APC Vio770 (clone REA805, Miltenyi Biotech).
Cells were fixed with fixation/permeabilization solution (BD Biosciences). For intracellular staining cell were permeabilized in Perm / wash solution (BD Biosciences) and stained with: IFN-a-PE Vio615 (clone REA1013, Miltenyi Biotec), TNF-a-APC (clone Mab11, Biolegend), and IP-10-PE (clone J034D6, Biolegend). Mouse cells were stained with the following antibodies: PDCA1-PE-Cyanine7 (clone eBio927, eBiosciences) and B220-APC-Cyanine7 (clone RA3-6B2, Invitrogen). For intracellular staining, cell were permeabilized in Perm/wash solution (BD Biosciences) and stained with : anti-IFNa-APC (clone RMMA-1, Invitrogen), anti-TNFa-PE (clone MP6-XT22, BD Biosciences), and anti-IL-12p40-PE-Cy5.5 (clone C17.8, Invitrogen). Samples were acquired on LSR II or Fortessa (BD Biosciences). The gating strategy is displayed as Supporting Information Fig. S4 and Fig. S5.
Cells were fixed with fixation/permeabilization solution (BD Biosciences). For intracellular staining cell were permeabilized in Perm / wash solution (BD Biosciences) and stained with: IFN-a-PE Vio615 (clone REA1013, Miltenyi Biotec), TNF-a-APC (clone Mab11, Biolegend), and IP-10-PE (clone J034D6, Biolegend). Mouse cells were stained with the following antibodies: PDCA1-PE-Cyanine7 (clone eBio927, eBiosciences) and B220-APC-Cyanine7 (clone RA3-6B2, Invitrogen). For intracellular staining, cell were permeabilized in Perm/wash solution (BD Biosciences) and stained with : anti-IFNa-APC (clone RMMA-1, Invitrogen), anti-TNFa-PE (clone MP6-XT22, BD Biosciences), and anti-IL-12p40-PE-Cy5.5 (clone C17.8, Invitrogen). Samples were acquired on LSR II or Fortessa (BD Biosciences). The gating strategy is displayed as Supporting Information Fig. S4 and Fig. S5.
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