Cryopreserved PBMC from 3 HUU at birth were thawed and counted as described above. Cells were stimulated for 24h in complete RPMI+10% FBS with TLR cocktail in duplicate. BFA (5ug/mL) was added to one replicate at the beginning of the stimulation, or for the last 4 hours of culture to the second replicate. Cells were washed with PBS, stained with Zombie Yellow Fixable Viability dye and surface stained with HLA-DR Alexa Fluor 488, PD-L1 BV421, CD19 PerCP-Cy5.5, CD69 APC-Cy7 (Biolegend), CD56 PE, CD16 PE-CF594, CD14 PerCP-Cy5.5, CD20 PerCP-Cy5.5 and CD3 Alexa Fluor700 (BD Biosciences). Cells were washed and treated with BD lysing solution and BD permeabilization buffer 2 (BD Biosciences) then stained for IL-8 PE-Cy7 (Biolegend) and IFNγ APC (eBiosciences). Cells were washed and fixed with PBS+1% paraformaldehyde before acquisition on the cytometer (Beckman Coulter Gallios).
Pd l1 bv421
Manufactured by BioLegend
PD-L1-BV421 is a fluorochrome-conjugated antibody that binds to the programmed cell death-1 ligand 1 (PD-L1) protein. It can be used for the detection and analysis of PD-L1 expression on cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd56 pe, by BD (4 mentions)
Celltrace violet, by Thermo Fisher Scientific (3 mentions)
Cd11c apc, by Miltenyi Biotec (3 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (3 mentions)
Dcp mm medium, by MatTek (3 mentions)
Cd3 pe fitc apc, by BD (3 mentions)
Cd8 apc fitc, by BD (3 mentions)
Cd123 pe pe cy5 fitc, by BD (3 mentions)
Cd138 fitc pe apc, by BD (3 mentions)
Bdca 2 fitc, by Miltenyi Biotec (3 mentions)
Cd3 alexa fluor 700, by BD (2 mentions)
Cd40 apc, by BioLegend (2 mentions)
Cd11a pe, by BioLegend (2 mentions)
Cd14 pe cy7, by BioLegend (2 mentions)
Cd80 pe, by BioLegend (2 mentions)
Mhcii fitc, by BioLegend (2 mentions)
Cd86 apc cy7, by BioLegend (2 mentions)
Tlr2 apc, by BioLegend (2 mentions)
Mrc1 pe, by BioLegend (2 mentions)
Siglec1 fitc, by BioLegend (2 mentions)
10 protocols using pd l1 bv421
1
Cryopreserved PBMC Immune Profiling
2
Immunophenotyping of Immune Cells
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For all experiments, cells were lifted by gentle scrapping, washed with PBS, and stained with MHCII-FITC (BioLegend, Cat no. 107606), CD40-APC (BioLegend, Cat no. 124612), PDL1-BV421 (BioLegend, Cat no. 124315), CD80-PE (BioLegend, Cat no. 104708), and CD86-APC-Cy7 (BioLegend, Cat no. 104708) TLR2-APC (Biolegend, Cat no. 153006) MRC1-PE (Biolegend, Cat no. 141706) Siglec1-FITC (Biolegend, Cat no. 142406) CD14-PE-Cy7 (Biolegend, Cat no. 123316) CD11a-PE (Biolegend, Cat no. 153103) (all diluted 1:400 in PBS). Cells were then washed 3 times in PBS and fixed with 1% formaldehyde (J.T. Baker, Cat no. JTB-2106-01) in PBS. Flow cytometry was performed on a BD LSR II or an Attune CytPix at the MSU Flow Cytometry Core, and data were analyzed using FlowJo (Version 10.8.1).
3
Investigating MM-pDC Interactions and Immunotherapies
4
Multiparametric Flow Cytometry of Murine Immune Cells
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Blood was taken routinely from anesthetized mice (retrobulbar venous plexus). Spleen and tumor tissues were dissociated. Single cells were stained with a panel of conjugated monoclonal antibodies (mAb, 0.125 μg to 1.5 μg each). Zombie NIR™ Fixable Viability Kit by Biolegend (San Diego, United States) staining was performed following the protocol Zombie NIR™ Fixable Viability Kit by Biolegend, extracellular staining was performed following the protocol BD Horizon Briliant Stain Buffer (BD Bioscience), followed by lysis and intracellular staining using the protocol of True-Nuclear™ Transcription Factor Buffer Set by Biolegend. Measurements were performed on a spectral flow cytometer (Cytek™ Aurora). For extracellular stainings Gr1 Alexa Fluor700, CD8 FITC, CD4 APC Fire, CD11b BV570, PD-L1 BV421, NK1.1 BV605, CD19 Spark Blue (Biolegend), CD25 PerCP-eFluor710 (Thermofisher), CD83 BV750, PD-1 BV650 (BD Bioscience) and for intracellular stainings CTLA-4 PE/Cy7, CD3 PerCP, and Foxp3 Alexa Fluor 647 (Biolegend) were used. Data were analyzed using SpectroFlow™ Version 2.2.0.3. and FlowJo™ Version 10.6.1.
5
Multiparameter Flow Cytometry Panel
6
Coculture of Myeloid Dendritic Cells
7
Multiparameter Flow Cytometry Panel
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Mouse anti-human CD11c-PE/Cy7, EPCAM-PE/CF594, CD86-BV510, HLA-DR PerCP/Cy5.5, PD-L1-BV421, PD-L2-APC, and CD83-APC were purchased from Biolegend (San Diego, CA). Fixable Viability Dye eFluor™ 780 was purchase from eBioscience (San Diego, CA) and mouse anti-human CD56-FITC were purchased from BD Biosciences (San Jose, CA). Recombinant human IL-4 and GM-CSF were purchased from R&D systems (Minneapolis, MN) reconstituted according manufacturer instructions and kept at −80 Celsius freezer in aliquots. 2′, 3′- cGAMP sodium salt was purchased from Tocris (Bristol, UK). Cetuximab (anti-EGFR mAb, IgG1) was kindly provided by Bristol-Meyers Squibb and was used at a concentration of 10 ug/mL in all our experiments. Human IgG1 Kappa-LE/AF was purchased from Sourthern Biotech (Birmingham, AL). Western blot antibodies included rabbit anti-human STING, pIRF3, and total IRF3 were purchased from Cell Signaling (Danvers, MA). Mouse anti-human β-actin was purchased from Bio Rad (Hercules, CA). Secondary goat anti-rabbit and anti-mouse antibodies were purchased from LI-COR biosciences (Lincoln, NE). STING primary IHC antibody was purchased from Abcam (Cambridge, MA). Human IFN-gamma Quantikine ELISA Kit was purchased from R&D systems (Minneapolis, MN).
8
Multicolor Flow Cytometry Panel
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The following anti-human antibodies were used for staining: CD68-FITC, CD14-PercP-Cy5.5, HLA-DR-APC, CD15-PE-Cy7, CD11b-PErcP-Cy5.5, CD163-BV421, CD14-APC, HLA-DR-PE, CD33-BV421 and PD-L1-BV421 purchased from Biolegend (San Diego, CA), CD11b-PE, CD3-Alexa Fluor 700, CD19-Alexa Fluor 700, CD19-Alexa Fluor 700, CD163-Alexa Fluor 647, CD16-PE-Cy7, CD3-PE, CD19-PE and CD56-PE purchased from BD Biosciences (San Jose, CA), CD14-PE-TR and CD16 PE-TR purchased from Life Technologies (Carlsbad, CA) and CD86-FITC purchased from R&D systems (Minneapolis, MN). CD32-a-FITC Ab was purchased from Stemcell technologies, (Vancouver, Canada). CD-32-B (F-4) was purchased from Santa Cruz Biotechnology, Santa Cruz, CA, and secondary Ab anti-mouse F(ab’)2 was purchased from Thermo fisher scientific (MA, USA).
Intracellular staining of CD68 was performed as follows: PBMC were stained with surface marker antibodies, fixed with fixation/permeabilization buffer (eBioscience), washed, and stained for intracellular antigens in 1X permeabilization buffer. Cells were analyzed on an LSR Fortessa (BD) flow cytometer, and data analyzed using Flow Jo (Treestar, Ashland, OR). Dead cells were excluded based on viability dye staining (Zombie Aqua Fixable Viability Dye, Biolegend).
Intracellular staining of CD68 was performed as follows: PBMC were stained with surface marker antibodies, fixed with fixation/permeabilization buffer (eBioscience), washed, and stained for intracellular antigens in 1X permeabilization buffer. Cells were analyzed on an LSR Fortessa (BD) flow cytometer, and data analyzed using Flow Jo (Treestar, Ashland, OR). Dead cells were excluded based on viability dye staining (Zombie Aqua Fixable Viability Dye, Biolegend).
9
Plasmacytoid Dendritic Cell Characterization
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MM-pDCs were co-cultured either in DCP-MM medium (Mattek Corp. Ashland, MA) or complete RPMI-1640 medium supplemented with IL-3 (Peprotech Inc., Rocky Hill, NJ, USA). CD3-PE/FITC/APC; CD4-FITC/PE or APC-Cy7; CD8-APC/FITC, CD56-PE; CD123-PE/PE-Cy5/FITC; and CD138-FITC/PE/APC were obtained from BD Biosciences (San Jose, CA). BDCA-2-FITC and CD11c-APC were obtained from Miltenyi Biotec (Auburn, CA); CD303-, CD304-, CD107a, CD138-BV421, and PD-L1-BV421 were purchased from Biolegend. Immunomagnetic separation kits were purchased from Miltenyi Biotec. The CellTrace Violet and CellTracker Violet/Green flow assay kits were obtained from Life Technologies (USA). CD73 blocking antibody (anti-human CD73 Abs) and TLR-7 agonist were obtained from eBiosciences and Invivogen, respectively. WST-1 Cell Proliferation Reagent was purchased from Clontech Laboratories, Inc. (USA).
10
Multiparametric Flow Cytometry Analysis
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For all experiments, the cells were lifted by gentle scraping, washed with PBS, and stained with MHC-II-FITC (BioLegend, catalog no. 107606), CD40-APC (BioLegend, catalog no. 124612), PDL1-BV421 (BioLegend, catalog no. 124315), CD80-PE (BioLegend, catalog no. 104708), CD86-APC-Cy7 (BioLegend, catalog no. 104708), TLR2-APC (BioLegend, catalog no. 153006), MRC1-PE (BioLegend, catalog no. 141706), Siglec1-FITC (BioLegend, catalog no. 142406), CD14-PE-Cy7 (BioLegend, catalog no. 123316), CD11a-PE (BioLegend, catalog no. 153103), and CD69-PE (BioLegend, catalog no. 104508) (all diluted 1:400 in PBS). The cells were then washed three times in PBS and fixed with 1% formaldehyde (J. T. Baker, catalog no. JTB-2106-01) in PBS. Flow cytometry was performed on a BD LSR II or an Attune CytPix at the Michigan State University Flow Cytometry Core, and the data were analyzed using FlowJo (version 10.8.1).
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