Cell phenotype was assessed by flow cytometry (BD LSRFortessa; BD Biosciences) after incubation with the following fluorescein-labeled antibodies: CD45-allophycocyanin (APC)-eFluor780, CD3e-fluorescein isothiocyanate (FITC), CD4-APC, CD8a-phycoerythrin (PE)-cy7, CD25-PE-cy7, Foxp3-PE, IFN-γ-AlexaFluor488, IL-4-PE-cy7, CD107a-PE, granzymeB-peridinin chlorophyll (PerCP)-eFluor710, perforin-FITC, CD11c-PE-cy7, CD80-PerCP-eFluor710, CD83-FITC, B7-H4-PE, and CD31-PE. All antibodies were purchased from eBioscience (San Diego, CA, USA), except CD4-APC, CD8a-PE-cy7, and Foxp3-PE (BD Biosciences). Intracellular antigens were determined after incubation with ionomycin (500 ng/mL; Abcam, Cambridge, UK) and phorbol-12-myristate13-acetate (10 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) for 1 h and monensin (2 μM; eBioscience) for an additional 4 h. Fixation and permeabilization were performed prior to antibody incubation, and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
Cd80 percp efluor710
Manufactured by BD
The CD80-PerCP-eFluor710 is a fluorochrome-conjugated monoclonal antibody that binds to the CD80 cell surface protein. CD80 is a co-stimulatory molecule expressed on antigen-presenting cells, such as dendritic cells and B cells, and plays a role in T cell activation. The PerCP-eFluor710 fluorescent label allows for the detection of CD80-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd31 pe, by BD (1 mentions)
Cd4 apc, by BD (1 mentions)
Cd25 pe cy7, by BD (1 mentions)
Cd11c pe cy7, by BD (1 mentions)
Cd8a pe cy7, by BD (1 mentions)
Foxp3 pe, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Cd83 fitc, by BD (1 mentions)
Cd107a pe, by BD (1 mentions)
B7 h4 pe, by BD (1 mentions)
Ionomycin, by Abcam (1 mentions)
Perforin fitc, by BD (1 mentions)
Il 4 pe cy7, by BD (1 mentions)
Ifn γ alexa fluor 488, by BD (1 mentions)
Cd3e fluorescein isothiocyanate fitc, by BD (1 mentions)
Cd86 pe cy7, by BD (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Humannaive cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
2 protocols using cd80 percp efluor710
1
Flow Cytometry Immunophenotyping Protocol
2
Differentiation and Activation of Human moDCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Peripheral blood
mononuclear cells were isolated
from buffy coats obtained from HLA-A2.1+ healthy volunteers
after written informed consent and in agreement with institutional
guidelines using Ficoll density gradient centrifugation (Lymphoprep,
ELITechGroup). CD14+ monocytes and naïve CD8+ T cells were isolated using CD14 microbeads and the human
Naive CD8+ T cell isolation kit (both Miltenyi Biotec),
respectively. Monocytes were differentiated into immature monocyte-derived
DCs (moDCs) in 6 days using RPMI 1640 containing 10% FCS, 2 mMl -glutamine, 0.5% antibiotic–antimycotic enriched with
interleukin-4 (IL-4, 300 U/mL), and GM-CSF (450 U/mL) (both Miltenyi
Biotec). Isolated cells were stored in liquid nitrogen in 10% DMSO
(CryoSure) and 90% FBS.
Human moDCs were thawed from liquid
nitrogen. In vitro DC activation (24 and 48 h) in
alginate cryogels containing NY-ESO-1 PLGA NPs was studied, as described
for BMDCs. Soluble NY-ESO-1 peptide and TLR ligands were added to
the controls in the same amounts as is present in NPs. Flow cytometric
staining was performed using Zombie Violet Fixable viability dye for
30 min at 4 °C, followed by cell-surface staining with the following
antibodies for 30 min at 4 °C: CD14-FITC (Miltenyi Biotec), CD80-PerCP-eFluor710
(PharMingen), CD86-PeCy7 (PharMingen), and CD40-PE (Beckman).
mononuclear cells were isolated
from buffy coats obtained from HLA-A2.1+ healthy volunteers
after written informed consent and in agreement with institutional
guidelines using Ficoll density gradient centrifugation (Lymphoprep,
ELITechGroup). CD14+ monocytes and naïve CD8+ T cells were isolated using CD14 microbeads and the human
Naive CD8+ T cell isolation kit (both Miltenyi Biotec),
respectively. Monocytes were differentiated into immature monocyte-derived
DCs (moDCs) in 6 days using RPMI 1640 containing 10% FCS, 2 mM
interleukin-4 (IL-4, 300 U/mL), and GM-CSF (450 U/mL) (both Miltenyi
Biotec). Isolated cells were stored in liquid nitrogen in 10% DMSO
(CryoSure) and 90% FBS.
Human moDCs were thawed from liquid
nitrogen. In vitro DC activation (24 and 48 h) in
alginate cryogels containing NY-ESO-1 PLGA NPs was studied, as described
for BMDCs. Soluble NY-ESO-1 peptide and TLR ligands were added to
the controls in the same amounts as is present in NPs. Flow cytometric
staining was performed using Zombie Violet Fixable viability dye for
30 min at 4 °C, followed by cell-surface staining with the following
antibodies for 30 min at 4 °C: CD14-FITC (Miltenyi Biotec), CD80-PerCP-eFluor710
(PharMingen), CD86-PeCy7 (PharMingen), and CD40-PE (Beckman).
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