Cells were incubated for 30 min on ice in 100 μl Hank’s balanced salt solution (Thermo Fisher) + 2% FBS with Fc block (1:100 dilution, clone 2.4G2; BD Biosciences) along with the following antibodies (all from BD Biosciences): CD45 APC-Cy7 (1:500, clone 30-F11), CD11b AF488 (1:500, clone M1/70), CD80 BV421 (1:200, clone 16-1OA1), and CD86 PE-Cy7 (1:500, clone GL1). IA/I-E AF647 (1:500, clone M5/114.15.2; BioLegend). Cells were then fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) according to manufacturer’s instructions. Cells were then incubated with an anti-RVFV antibody (kindly provided by Dr. Robert Tesh and the World Reference Center of Emerging Viruses and Arboviruses) at 1:500 dilution, followed by goat anti-mouse PE secondary antibody (1:1000, Santa Cruz biotechnology). Flow cytometry was performed using a FACSAria Fusion and data were analyzed using FlowJo software. Microglia, other myeloid lineage, and lymphocytes were resolved using CD45 and CD11b expression, with microglia identified as CD45int CD11bint, other myeloid as CD45hi CD11bhi, and lymphocytes as CD45hi CD11b– as previously described [65 (link)].
Cd80 bv421
Manufactured by BD
Sourced in United States
The CD80 BV421 is a fluorochrome-conjugated antibody reagent used in flow cytometry. It is designed to detect the expression of the CD80 (B7-1) surface antigen on cells. The CD80 BV421 is a tool for immunophenotyping and monitoring immune cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Fc block, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Cd86 pe cy7, by BD (1 mentions)
Cd45 apc cy7, by BD (1 mentions)
Cd11b af488, by BD (1 mentions)
Ia 1 e af647, by BioLegend (1 mentions)
Cd3 fitc, by BD (1 mentions)
Cd86 fitc, by BD (1 mentions)
B220 apc, by Thermo Fisher Scientific (1 mentions)
Cd206 alexa fluor 647, by BD (1 mentions)
Cd11b percp cy5, by BD (1 mentions)
Cd40 percp cy5, by Thermo Fisher Scientific (1 mentions)
Facs fortessa 2 flow cytometer, by BD (1 mentions)
Cd4 apc fire750, by BD (1 mentions)
F4 80 bv711, by BD (1 mentions)
Cd3 apc cy7, by BD (1 mentions)
Live dead fixable blue dye, by Thermo Fisher Scientific (1 mentions)
Cd14 bv510, by BioLegend (1 mentions)
Cd40 fitc, by BioLegend (1 mentions)
4 protocols using cd80 bv421
1
Quantifying Virus-Infected Immune Cells
2
Characterization of Murine Splenocytes
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Spleen cell suspensions were prepared after hypotonic lysis of erythrocytes in potassium acetate solution and three washes in complete RPMI medium. For each mouse, splenocytes were quantified using a Malassez counting chamber. Cells were then incubated with an antibody at 4 °C for 30 min in the dark in PBS with 2% normal FBS. Flow cytometry was performed using a FACS Fortessa II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), according to standard techniques. To characterize splenic cells, the monoclonal antibodies used spleen cell suspensions prepared after hypotonic lysis of erythrocytes in potassium acetate solution and three washes in complete RPMI medium. For each mouse, splenocytes were quantified using a Malassez counting chamber. Cells’ suspensions were incubated for 20 min with the following antibodies. All antibodies were obtained from Biolegend (San Diego, CA, USA) and used at a dilution of 1:100 unless otherwise mentioned: CD3 FITC, CD4 APC Fire 750 (BD Biosciences, San Jose, CA, USA), CD8 BV 605, CD69 PEDazzle594, CD40 PercPCy5.5, B220 APC, CD44 BV650, MHC II eFluor450 (DAPI) (eBioscience, San Diego, CA, USA), F4/80 BV711 (dilution 1:200), CD11b PercP Cy5.5, CD80 BV421, CD86 FITC, CD206 Alexa Fluor 647 (BD Biosciences, San Jose, CA, USA. Dilution 1:200), Ly6C PECy7.
3
Profiling Immune Cell Subsets Post-Vaccination
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On days 0, 1, and 14 after the first immunization, the cellular composition of the PBMCs was analyzed using flow cytometry. Freshly isolated PBMCs were stained with Live/Dead Fixable Blue Dye (Life Technologies, cat# L-23105, 1:40 dilution) and FcR blocking reagent (Miltenyi Biotec, cat# 130-059-901, 1:20 dilution) followed by a panel of antibodies: CD40 FITC (5C3, Biolegend, cat# 334306, 1:20 dilution), NKG2A PE (Z199, Beckman Coulter, cat# IM3291U, 1:40 dilution), CD80 BV421 (L307.4, BD, cat# 564160, 1:40 dilution), CCR7 PE-Dazzle594 (G043H7, Biolegend, cat# 353236, 1:50 dilution), CD123 Per-CP-Cy5.5 (7G3, BD, cat# 558714, 1:80 dilution), CD3 APC-Cy7 (SP34-2, BD, cat# 557757, 1:80 dilution), CD66 APC (TET2, Miltenyi Biotec, cat# 130-118-539, 1:80 dilution), CD70 BV786 (Ki-24, BD, cat# 565338, 1:80 dilution), HLA-DR BV650 (L243, Biolegend, cat# 307650, 1:80 dilution), CD11c PE-Cy7 (3.9, Biolegend, cat# 301608, 1:160 dilution), CD16 AF700 (38 G, BD, cat# 560713, 1:160 dilution), CD20 BV605 (2H7, Biolegend, cat# 302334, 1:160 dilution) and CD14 BV510 (M5E2, Biolegend, cat# 301842, 1:160 dilution). After washing with PBS, samples were fixed using 1% paraformaldehyde (PFA) and acquired on a BD LSRFortessa cell analyzer. The data were analyzed using FlowJo software v.10.7.1 (FlowJo).
4
Generating Mature Dendritic Cells
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Peripheral blood mononuclear cells (PBMCs) from human healthy donors were thawed from ImmunXperts SA (Belgium) biobank. Monocytes were isolated from PBMCs using a MACS magnetic separation column CD14 MicroBeads (Miltenyi) and purity was evaluated by CD14 FACS staining (Fortessa). Cells were then resuspended at a cellular density of 106 cells/mL and plated into a 24-well tissue culture microplate (1mL per well) in CellGenix DC medium (CellGenix, Cat.N° 20801-0500) added with Gentamycin, IL-4 (Miltenyi, 130-093-866) and GM-CSF (Miltenyi, 130-093-922) for 5 days. At day 5, cells were stained for FACS analysis with several DC activation markers to assess their immature dendritic cell (iDC) state: CD14-FITC (Miltenyi, 130-110-518), CD40-BV510 (BD Biosciences, 563456), CD80-BV421 (BD Biosciences, 564160), CD83-PE-Vio 770 (Miltenyi, 130-110-505), CD86-APC (Miltenyi, 130-116-161), CD209-PE (Miltenyi, 130-117-706), and HLA-DR-BUV395 (BD Biosciences, 564040). On the same day, respective antigens (10µg/mL) were added to the cell culture for 48h. At day 7, cells were stained for FACS analysis with same markers to assess their mature state. LPS (Sigma, L4391-1MG) (1µg/mL), and TNF (Miltenyi, 130-094-024) (800U/mL), with IL-1b (Miltenyi, 130-093-898) (150U/mL), were used as positive control.
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