HLA-DRA and HLA-DRB chains were produced as previously described (42 (link)). For tetramer production, HLA-DRA and HLA-DRB chains were mixed in a 1:1.5 ratio and incubated in 3 μM peptides in PBS (pH 7.4) with 20% glycerol and 0.1% Pluronic F68 for 96 h at 18°C. The resulting monomers were buffer changed into PBS with 5% glycerol and concentrated on 10kD Vivaspin (Satorius) and quantitated by Luminescent Oxygen Channeling Immunoassay (LOCI)-driven assay (42 (link)). The resulting monomers were tetramerized by addition of fluorochrome labeled streptavidin (streptavidin-phycoerythrin (SA-PE) or streptavidin-allophycocyanin (SA-APC); both from BioLegend) sequentially over 60 min at a 1:4 molar ratio of streptavidin to monomer. For T cell analysis, pellets of 4 × 105 cells obtained from in vitro peptide stimulated cell cultures, were re-suspended in a 40 μl tetramer diluted in media to a final concentration of ca. 30 nM, and incubated for 1 h at 37°C, followed by 30 min incubation with fluorochrome conjugated anti-CD3, -CD8, -CD38, and -HLA-DR antibodies. The cells were analyzed by flowcytometry (Fortessa or LSR-II, BD Biosciences) using Diva software.
Streptavidin streptavidin phycoerythrin sa pe
Manufactured by BioLegend
Streptavidin-phycoerythrin (SA-PE) is a conjugate of the protein streptavidin and the fluorescent dye phycoerythrin. Streptavidin has a high affinity for the small molecule biotin, which allows it to be used as a detection agent in various biological assays.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using streptavidin streptavidin phycoerythrin sa pe
1
HLA-DRA/DRB Tetramer Production and T Cell Analysis
2
HLA Tetramer Production and T Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HLA-DRA and HLA-DRB chains were produced as previously described (43) (link). For tetramer production, HLA-DRA and HLA-DRB chains were mixed in a 1:1.5 ratio and incubated in 3 µM peptides in PBS (pH 7.4) with 20% glycerol and 0.1% Pluronic F68 for 96h at 18⁰C. The resulting monomers were buffer changed into PBS with 5% glycerol and concentrated on 10kD Vivaspin (Satorius) and quantitated by Luminescent Oxygen Channeling Immunoassay (LOCI)-driven assay (43) (link). The resulting monomers were tetramerized by addition of fluorochrome labelled streptavidin (streptavidin-phycoerythrin (SA-PE) or streptavidin-allophycocyanin (SA-APC); both from BioLegend) sequentially over 60 min at a 1:4 molar ratio of streptavidin to monomer. For T cell analysis, pellets of 4x10 5 cells obtained from in vitro peptide stimulated cell cultures, were resuspended in a 40 µl tetramer diluted in media to a final concentration of ≈ 30 nM, and incubated for 1h at 37°C, followed by 30 min incubation with fluorochrome conjugated anti-CD3, -CD8, -CD38 and -HLA-DR antibodies. The cells were analyzed by flowcytometry (Fortessa or LSR-II, BD Biosciences) using Diva software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!