To examine the expression level of molecules on the cell surface, cells harvested after the MLR were washed with PBS containing 2% FBS and then stained with the following antibodies (Abs): phycoerythrin-cychrome 5 (PC5)-conjugated anti-CD8 (Beckman Coulter, Inc., Brea, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated anti-CD3, phycoerythrin (PE)-conjugated anti-CD4, FITC-conjugated anti-CD25 (Becton Dickinson, Franklin Lakes, NJ, USA), PE-conjugated anti-CD45RA, or PE-conjugated anti-CD45RO (BioLegend, San Diego, CA, USA) at room temperature in the dark for 30 min. Cells were then washed with PBS containing 2% FBS and resuspended in 0.3 ml of PBS containing 2% FBS for analysis by flow cytometry (FCM) using FACS CaliburTM (Becton Dickinson) as previously described [14 ].
Pe conjugated anti cd45ro
Manufactured by BioLegend
Sourced in United States
PE-conjugated anti-CD45RO is a monoclonal antibody that binds to the CD45RO antigen expressed on the surface of certain human immune cells. The PE (Phycoerythrin) fluorescent dye is conjugated to the antibody, enabling detection and analysis of CD45RO-positive cells using flow cytometry.
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Lab products found in correlation
Fitc conjugated anti cd25, by BD (2 mentions)
Facscalibur, by BD (1 mentions)
Fitc conjugated anti cd3, by BD (1 mentions)
Apc conjugated anti il 17a, by BioLegend (1 mentions)
Pacific blue conjugated anti cd4, by BioLegend (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti ifn γ, by BD (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
4 protocols using pe conjugated anti cd45ro
1
Cell Surface Molecule Expression Analysis
2
T Cell Phenotype Analysis
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After one week of culture with indicated treatments, PBMCs were analyzed for their central memory, effector memory or naive T cell phenotype, by incubation with PE-conjugated anti-CD45RO, FITC-conjugated anti-CCR7, and APC-conjugated anti-CD3 antibodies (Biolegend). Isotype-matched non-specific antibodies were used as negative controls. Staining was analyzed on viable cells on forward scatter/sideward scatter plot, and on the CD3+ population gate.
3
Multiparametric Analysis of Immune Cells
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PBMCs and SFMCs were isolated and suspended in a complete medium (RPMI 1640, 2 mM L-glutamine, 100 units/ml of penicillin, and 100 μg/ml of streptomycin) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA), and then seeded into 96-well plates at cell density of 1 × 106 cells/well. Cells in a 96-well culture plate were treated with CSp and then were activated with Dynabeads Human T-Activator CD3/CD28 (11131D, Gibco, USA) to obtain a bead to cell ratio of 1:1. Cells were then incubated in a humidified CO2 incubator at 37°C for 24 h. After stimulating with PMA (100 ng/ml) and ionomycin (1 μM) for 4 h, cells were stained with Pacific Blue-conjugated anti-CD4 (300521, BioLegend, USA), and PE-conjugated anti-CD45RO (304205, BioLegend, USA). Cells were washed, fixed, permeabilized with Cytofix/Cytoperm buffer, and stained intracellularly with FITC-conjugated anti-IFN-γ (552887, BD, USA), APC-conjugated anti-IL-17A (512334, BioLegend, USA) antibodies followed by analysis with FlowJo Software (BD, USA). In ex vivo cultured supernatants from PBMCs, INF-γ, IL-17A, TNF-α, and IL-6 were measured using ELISA (88-7316, 88-7176, 88-7346, and 88-7066, Invitrogen, Austria). The OD was recorded by a SpectraMax® M2(Molecular Devices Corp., USA) set at 450 nm.
4
Cell Surface and Intracellular Protein Expression
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To examine the expression level of molecules on the cell surface, cells harvested after the MLRs were washed with PBS containing 2% FBS and then stained with the following Abs: PC5-conjugated anti-CD8 and fluorescein isothiocyanate- (FITC-) conjugated anti-CD3, FITC-conjugated anti-CD25 (Becton Dickinson), phycoerythrin- (PE-) conjugated anti-CD45RA, or PE-conjugated anti-CD45RO (BioLegend, San Diego, CA, USA) at room temperature in the dark for 30 min. Cells were then washed with PBS containing 2% FBS and resuspended in 0.3 mL of PBS containing 2% FBS for FCM analysis. To examine the expression level of intracellular granzyme B, cells were harvested after the MLRs and surfaces were stained with PC5-conjugated anti-CD8 Ab as described above. Surface stained cells were washed with PBS containing 2% FBS and then fixed with 3.7% formaldehyde for 15 min. Fixed cells were washed with PBS containing 2% FBS. Fixed cells were permeabilized with 0.1% Triton 100 and stained with R-phycoerythrin- (RPE-) conjugated anti-granzyme B Ab (AbD Serotec, Oxford, UK) at room temperature in the dark for 30 min. Cells were then washed and resuspended as described above. The percentage of cells positive for each parameter was analyzed using FCM. Four independent experiments were performed.
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