Standard extracellular staining (20 mins at 24°C) was performed using the following fluorophore-labeled antibodies: CD3-BUV737 (BD Biosciences), CD4-BUV395 (BD Biosciences), CD8-BV711 (BD Biosciences), CD14-BV510 (BD Biosciences), CD19-BV510 (BioLegend), Live/Dead Fixable Aqua (ThermoFisher), CCR7-PE-CF594 (BD Biosciences), CD45RA-APC-H7 (BD Biosciences), CD28-APC-R700 (BD Biosciences), CD57-PE (BioLegend), PD-1-BV605, BV421 (BioLegend), CD69-BV650 (BioLegend), CD38-BV421 (BioLegend), HLA-DR-PerCP-Cy5.5 (BD Biosciences), TIGIT-PE-Cy7 (BioLegend), DNAM-BB515 (BD Biosciences), FcγRIIB-BV786 (BD Biosciences). Fluorescence minus one (FMO) controls were used to determine negative expression of FcγRIIB. Apoptosis was measured with Caspase-3/7 Green Flow Cytometry Assay Kit (ThermoFisher) or FITC Annexin V with 7-AAD Viability Staining Solution (BioLegend), according to the manufacturer’s instructions.
Pd 1 bv605
Manufactured by BioLegend
PD-1-BV605 is a monoclonal antibody that binds to the programmed cell death-1 (PD-1) receptor. PD-1 is an immunoinhibitory receptor expressed on activated T cells, B cells, and myeloid cells. The BV605 fluorochrome is conjugated to the PD-1 antibody.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 apc cy7, by BioLegend (3 mentions)
Cd8 bv510, by BioLegend (2 mentions)
Cd11c apc cy7, by BioLegend (2 mentions)
Cd3 bv510, by BioLegend (2 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (1 mentions)
Cd45ra apc h7, by BD (1 mentions)
Ccr7 pe cf594, by BD (1 mentions)
Cd8 bv711, by BD (1 mentions)
Cd14 bv510, by BD (1 mentions)
Cd19 bv510, by BioLegend (1 mentions)
Cd57 pe, by BioLegend (1 mentions)
Cd69 bv650, by BioLegend (1 mentions)
Cd38 bv421, by BioLegend (1 mentions)
Tigit pe cy7, by BioLegend (1 mentions)
Dnam bb515, by BD (1 mentions)
Caspase 3 7 green flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Cd4 buv395, by BD (1 mentions)
Cd3 buv737, by BD (1 mentions)
Hla dr percp cy5, by BD (1 mentions)
Mouse regulatory t cell staining kit 2, by Thermo Fisher Scientific (1 mentions)
9 protocols using pd 1 bv605
1
Multiparameter Flow Cytometry Phenotyping
2
Multiparametric Flow Cytometry of Tumor Cells
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Single-cell suspensions of tumors were prepared, and flow cytometry was carried out as mentioned earlier [45 (link), 46 (link)]. Cells were stained with the following antibodies obtained from BioLegend: CD16/32 (clone 93, catalog No. 103132), Zombie UV™ Fixable Viability Kit (catalog No. 100752), CD45-PerCP/Cy5.5 (clone 30-F11, catalog No. 103132), CD45-APC/Cy7 (clone 30-F11, catalog No. 103116), CD11b-FITC (clone M1/70, catalog No. 101206), SIRPα-PE (clone BM8, catalog No. 123122), Gr-1-PerCP/Cy5.5 (clone RB6-8C5, catalog No. 108428), F4/80-AF647 (clone N418, catalog No. 117318),CD11c-PE/Cy7 (clone N418, catalog No. 117318), I-A/I-E-AF700 (clone M5/114.15.2, catalog No. 107622), CD86-BV421 (clone GL-1, catalog No. 105032), CD206-BV605 (clone C068C2, catalog No. 141721), CD8a-AF700 (clone 53-6.7, catalog No. 100730), CD8-BV510 (clone 53-6.7, catalog No. 100752), TNF-α-BV421 (clone MP6-XT22, catalog No. 506328), IFN-γ-PE (clone XMG1.2, catalog No. 505808), CD39-PE/Cy7 (clone Duha59, catalog No. 143806), Tim-3-APC/Fire™ 750 (clone RMT3-23, catalog No. 119738), PD-1-BV605 (clone 29 F.1A12, catalog No. 135220). The next antibodies were obtained through eBioscience: Mouse Regulatory T Cell Staining Kit #2 (clone FJK-16s, catalog No. 88-8118-40). FlowJo program (Tree Star Inc.) was utilized to examine the findings that were acquired through a BD LSRFortessa Flow Cytometer.
3
Multiparameter Flow Cytometry of GC B Cells
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One million cells were stained in PBS+1% FBS. Cocktails of antibodies against the following surface proteins were used: B220 PerCP-Cy5.5 (RA3-6B2), B220 FITC (RA3-6B2), GL7 BV421 (GL7), IgD BV786 (11-26c.2a), Ly6G PE (1A8), CD11b PerCP-Cy5.5 (M1/70), CD11b PE-Cy7 (M1/70), CD4 APC (RM4-5), CxCR5 BV421 (2G8), CD11c BV421 (HL3) (All BD) CD38 PE-Cy7 (90), F4/80 PE-Cy7 (BM8), F4/80 APC-EF780 (eBioscience), CD11c APC-Cy7 (N418), and PD-1 BV605 (29F.1A12) (Biolegend). A live/dead marker was used to exclude dead cells in the GC B and TFH cell panels (Fixable Viability Dye eFluor™ 780, eBioscience). AF647-labeled ovalbumin (OVA-AF647) was from Invitrogen. Antigen-specific germinal center B cells were measured by including CTH522 coupled to AF488 as probe (conjugated by Life technologies at a coupling ratio of 3 moles dye/mole). Cells were analyzed on a BD Fortessa or FACSCanto flow cytometer.
4
Multiparametric Flow Cytometry Analysis
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Human HER2 and PD-L1 expression on tumor cells was detected using human HER2-PE-CY7 (clone 24D2, Biolegend) and PD-L1-APC (clone MIH1, eBioscience). Human T cells surface phenotype and transduction efficiency were assessed using the following antibodies: NGFR-FITC (clone ME20.4, Biolegend), Q8 (clone QBEND/10, ThermoFisher), CD45-AF700 or CD45-BV605 (clone HI30, Biolegend), CD3-APC (clone SK7, Biolegend), CD4-PerCP (clone SK3, Biolegend), CD4-BB700 (clone SK3, BD Bioscience), CD8-BV510 (clone SK1, Biolegend), CD27-PE-CY7 or BV786 (clone M-T271, Biolegend), CD28-BV605 or BV711 (clone CD28.2, Biolegend). Expression of T cell inhibitory receptors was analyzed using PD-1-BV421 or PD-1-BV605 (clone EH12.2H7, Biolegend), TIM-3-BV605 or TIM-3-PE-CY-7 (clone F38-2E2, Biolegend), CD39 (clone A1, Biolegend), LAG-3-BV711 (clone 11C3C65, Biolegend). Live/dead discrimination was determined using LIVE/DEAD fixable Near-IR dead cell stain kit (ThermoFisher). Mouse cells were assessed using the following antibodies: Flow cytometry results were analyzed using Kaluza software (Beckman Coulter).
5
Profiling T Cell Subsets in BAL Samples
6
Quantifying B7-H3 Expression in Tumor Cells
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B7-H3 expression levels on the surface of human tumor cells were detected using purified mAb-J42 or J42-scFv-Fc. Goat anti-mouse IgG–FITC (Proteintech) was used to label the Fc of mAb-J42. The antibodies used to identify the phenotype of CAR-T cells included CD3-allophycocyanin (APC)-CY7, perforin-APC, CD4-PE, CD8-FITC, TIM3-BV711, and PD-1-BV605 (all purchased from BioLegend). Flow cytometry analysis was performed on a BD Fortessa flow cytometer and analyzed using FlowJo 10.6.0 software.
7
Molecular Analysis of Cell Signaling Pathways
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Dulbecco’s Modified Eagle Medium (DMEM/HEPES, Cat #12430-054) and Penicillin/Streptomycin (Cat #15240062) were from Gibco (Carlsbad, CA). Fetal bovine serum (FBS) and G418 sulfate were obtained from Invitrogen (Carlsbad, CA). Rabbit antibody against β-actin (Cat #4970S) was from Cell Signaling (Beverly, MA). Rabbit antibody against NHE1 (Cat #ab67314) and rat antibody against CD8 (Cat #ab22378) were from Abcam Ltd. (Cambridge, MA). Rat anti-mouse CD31 (Cat #550274) was from BD Pharmingen (San Jose, CA). Rabbit anti-ionized calcium-binding adapter molecule 1 (iba1) was from Wako (Richmond, VA). PerCP/Cy5.5-CD45, PE-P2RY12, BV421-TGFβ, BV605-TNFα, APC/780-IL-1β, PE/Cy7-IL10, PerCP/Cy5.5-CD8a, APC/Cy7-CD4, Alexa Fluor 700-CD25, PE-FoxP3, BV605-PD-1, PE-Gr-1, APC-NK1.1, and Pacific Blue Granzyme B were obtained from Biolegend (San Diego, CA). eFluor 450-CD16/32, PE/Cy7-CD206, APC-IFNγ was purchased from eBioscience (San Diego, CA). APC-IL-6 were obtained from thermos fisher (Waltham, MA). BUV737-CD11b, Alexa Flour 700-CD86 were obtained from BD Biosciences (San Jose, CA). PE-Ym1were from Abcam Ltd. (Cambridge, MA). Rabbit anti-Laminin (Cat #L9393) and cariporide (HOE642) was purchased from Sigma Chemicals (St. Louis, MO). Anti-mouse PD-1 (RMP1-14) and IgG2a isotype control were from BioXcell (West Lebanon, NH).
8
Multiparametric Flow Cytometry Analysis of Murine Splenocytes
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An LSR Fortessa (BD Biosciences, Franklin Lakes, NJ) was used for multiparametric flow cytometry analysis. A Live/Dead Fixable Blue Dead Cell Stain Kit (Molecular Probes; Thermo Fisher Scientific, Grand Island, NY) was used to exclude dead cells. The following murine monoclonal antibodies (mAbs) were used to stain Balb/c splenocytes to characterize immune cell subsets: FITC-CD8 (Clone 53-6.7; BD Biosciences), PE-CD49b (Clone DX5; BioLegend, San Diego, CA), PE-PD-L1 (Clone 10F.9G2; BioLegend), PerCP-Cy5.5-B7-1 (Clone 16-10A1; BioLegend), PerCP-Cy5.5-FOXP3 (Clone FJK-16s; eBioscience, San Diego, CA), PE-Cy7-CD122 (Clone Tm-b1; eBioscience), APC-NKG2D (Clone CX5; BioLegend), APC-Cy7-CD3 (Clone 17A2; BioLegend), Pacific Blue-CD44 (Clone IM7; BioLegend), AF700-CD4 (Clone GK1.5; eBioscience), BV605-CD19 (Clone 6D5; BioLegend), BV605-PD-1 (Clone 29F.1A12; BioLegend), BV510-NKp46 (Clone 29A1.4; BioLegend), Pacific Blue-CD27 (Clone LG.3A10; BioLegend), FITC-CD11b (Clone M1/70; BD Biosciences), AF700-CD11b (Clone M1/70; BioLegend), APC-Cy7-CD11c (Clone N418; BioLegend), BV510-CD3 (Clone 17A2; BioLegend), Pacific Blue-Ly6-G (Clone 1A8; BioLegend), BV605-Ly6-C (Clone HK1.4; BioLegend).
9
Tumor Immune Cell Profiling by Flow Cytometry
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Tumors were processed for flow cytometry into single cells using mechanical and enzymatic digestion (enzyme mixture—collagenase, DNAse, and hyaluronidase). Red blood cells were lysed as described above and viable tumor cells counted. The following antibodies were used to stain immune cell populations: Ghost violet 510 Live/Dead, Pacific Blue CD45, PE-594 CD3, PE/Cy7 CD8, APC/Cy7 CD4, BV605 PD-1, APC Tim3, FITC CD62L, BV711 CD44 (BioLegend).
Samples were run on an LSR Fortessa machine. Single color compensation controls were performed using compensation beads (Thermo Fisher Scientific, Waltham, MA, USA) to correct for overlap in signal among antibodies. Spleen samples were used to set a gating strategy for CD3+/CD4+ and CD3+/CD8+ T cells. FlowJo software (version 10) was used to perform all downstream analyses on subpopulations.
Samples were run on an LSR Fortessa machine. Single color compensation controls were performed using compensation beads (Thermo Fisher Scientific, Waltham, MA, USA) to correct for overlap in signal among antibodies. Spleen samples were used to set a gating strategy for CD3+/CD4+ and CD3+/CD8+ T cells. FlowJo software (version 10) was used to perform all downstream analyses on subpopulations.
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