PBMCs were isolated over Ficoll and cryopreserved. Cells were rapidly thawed in warmed Roswell Park Memorial Institute medium/10% fetal calf serum (FCS), before viability staining with Violet LIVE/DEAD Fixable stain kit (VIVID, Invitrogen, Paisley, United Kingdom), then washed and resuspended for incubation with either α-galcer–loaded CD1d tetramer or control CD1d tetramer (Proimmune, Oxford, United Kingdom) for 30 minutes on ice, protected from light. Subsequently, cells were washed, stained with antibody mastermix 1 (Supplementary Table 1 ) for 30 minutes at 4°C, washed and resuspended in phosphate-buffered saline, 1% Hi-FCS, and 2% paraformaldehyde, then incubated for 1 hour, washed, and resuspended for acquisition.
α galcer loaded cd1d tetramer
Manufactured by ProImmune
Sourced in United Kingdom, Canada
The α-galcer–loaded CD1d tetramer is a laboratory tool used in immunological research. It consists of multiple copies of the CD1d protein, each loaded with the α-galactosylceramide (α-galcer) ligand, and assembled into a tetrameric structure. This product allows for the identification and study of natural killer T cells (NKT cells) that express T cell receptors specific for the α-galcer-CD1d complex.
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Anti pd 1, by BioLegend (1 mentions)
Pb conjugated anti cd4, by BD (1 mentions)
Pe cy7 conjugated anti cd8, by BD (1 mentions)
Anti ccr7, by BioLegend (1 mentions)
Allophycocyanin conjugated anti cd3, by BD (1 mentions)
2 protocols using α galcer loaded cd1d tetramer
1
Cryopreserved PBMC Phenotyping
2
Longitudinal Immunophenotyping of PBMC
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PBMC samples were obtained from patients before the administration of αGalCer-pulsed APCs, and another eight times during the 11-week study period (figure 1 ). mAbs to detect and fractionate mononuclear cells in peripheral blood were: FITC-conjugated anti-TCR Vα24 (Beckman Coulter, Brea, CA), anti-CD14 (BD Biosciences), anti-CD45RA (BD Biosciences), PE-conjugated anti-TCR Vβ11 (Beckman Coulter), anti-CD56 (BD Biosciences), anti-CCR7 (Biolegend), allophycocyanin-conjugated anti-CD3 (BD Biosciences), αGalCer-loaded CD1d tetramer (ProImmune), allophycocyanin-Cy7 conjugated anti-CD3 (BD Biosciences), PE-Cy7-conjugated anti-CD8 (BD Biosciences), PB-conjugated anti-CD4 (BD Biosciences), anti-CD16 (BD Biosciences), anti-PD-1 (Biolegend), and anti-HLA-DR(Biolegend). The absolute numbers of these cells were also calculated using automated full blood counts.
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