Isolated splenocytes (1 × 106 cells/well) were stimulated for 1 h with 5 µg/ml MOMP in the presence of 1 µg/ml anti-CD28 and anti-CD49d mAbs (both BD Pharmingen, San Diego, CA, USA) in a total volume of 200 µl supplemented with RPMI 1640. The cells were subsequently incubated for 5 h at 37℃ after addition of 10 µg/ml brefeldin A (Sigma Aldrich) and 0.7 µl/ml monensin/Golgi-stop (BD Pharmingen). Following overnight storage at 4℃, the cells were washed in FACS buffer and stained with anti-CD4 (APC) and anti-CD44 (FITC) mAbs (BD Biosciences) in FACS buffer for 30 min at 4℃. Cells were washed with FACS buffer, permeabilized by using the Cyto-fix/Cyto-perm kit (BD Pharmingen) according to the manufacturer’s instructions, and stained intracellularly for 30 min at 4℃ using anti-IFN-γ (PE–Cy7) and anti-IL-17a (PerCP–Cy5.5) mAbs (eBiosciences, San Diego, CA, USA) in Perm wash buffer. After washing, the cells were re-suspended in FACS buffer and analysed by flow cytometry using a six-colour BD FACS Canto flow cytometer (BD Biosciences). Responses were analysed with the FlowJo software (Tree Star Inc., Ashland, OR, USA).
Anti cd28 and anti cd49d mabs
Manufactured by BD
Sourced in United States
Anti-CD28 and anti-CD49d monoclonal antibodies (mAbs) are laboratory reagents used in flow cytometry and other immunological applications. These antibodies can bind to specific cell surface markers, CD28 and CD49d, which are expressed on various immune cell types. The core function of these mAbs is to enable the detection, identification, and analysis of cells expressing the target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Monensin golgi stop, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Anti ifn γ pe cy7, by Thermo Fisher Scientific (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Cell activation cocktail, by BioLegend (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
3 protocols using anti cd28 and anti cd49d mabs
1
Flow Cytometric Analysis of Cytokine Production
2
T cell activation assay for COVID-19
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To measure T cell activation, PBMCs were stimulated with the commercially available cell activation cocktail (Biolegend) containing phorbol 12-myristate-13-acetate (PMA) and ionomycin in the presence of brefeldin A (BFA, 7.5 μg/mL, Sigma-Aldrich) for 6 h. For COVID-19-specific T cell responses, PBMCs were stimulated with 1 μg/mL COVID-19 RBD peptide pool (15-mer overlapping by 11, spanning the whole RBD sequence at Spike306-543) or 5 μg/mL purified nucleocapsid (NP) protein in the presence of 0.5 μg/mL anti-CD28 and anti-CD49d mAbs (BD Bioscience). Cells were incubated at 37°C overnight and BFA was added at 2 h post incubation, as previously described (Li et al., 2008a (link)). PMA/ionomycin stimulation was included as positive control. After overnight incubation, cells were washed with staining buffer (PBS containing 2% FBS) and stained with mAbs against surface markers. For intracellular staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) prior to staining with the mAbs against cytokines (Table S2 ) with Pern/Wash buffer (BD Biosciences). Results were considered positive when there was at least a 2-fold increase above the background of HD.
3
Alloactivation Analysis in MLR Assay
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CFSE-labeled responder cells (R) derived from CB or APB were plated in triplicate in 96-well round bottom microtiter plates at a density of 1 × 10 5 cells per well, and irradiated HLA-mismatched stimulator cells (S) were added to the co-culture system at a ratio of 1:1 (S/R). Phytohemagglutinin (PHA, 5 μg/ml) (Sigma, St. Louis, MO)-stimulated responder cells were used as the positive control, and responder cells stimulated by mitomycin-treated autologous MNCs were used as the negative control. Co-stimulation was provided by the addition of anti-CD28 and anti-CD49d mAbs (BD Fast Immune, San Jose, CA) both at 1 μg/ml to the MLR or control cultures. Mixed lymphocytes were cultured in a final volume of 200 μL RPMI 1640 supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 10% heat-inactivated FBS, and placed at 37°C under 5% CO 2 humidified atmosphere.
For alloactivation analysis, intracellular antigen staining and CD25 + ATs isolation, co-cultures were harvested on day 5, at which time point the expression of CD25 reached a peak level, according to our sequential observations and others' experiences (Amrolia et al. 2003) . On day 8, the remaining cultures were harvested for cell division analysis with CFSE dilution method, when the maximum proliferation was observed in our MLR system.
For alloactivation analysis, intracellular antigen staining and CD25 + ATs isolation, co-cultures were harvested on day 5, at which time point the expression of CD25 reached a peak level, according to our sequential observations and others' experiences (Amrolia et al. 2003) . On day 8, the remaining cultures were harvested for cell division analysis with CFSE dilution method, when the maximum proliferation was observed in our MLR system.
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