TITLE: Flex-T™ Tetramer and Cell Staining Protocol
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Using UV-induced peptide exchange, MHC/peptide monomers can be generated with conditional Flex-T™ monomers that harbor peptides of interest in their binding grooves. These new MHC monomers are subsequently multimerized using streptavidin-fluorophore conjugates. The resulting Flex-T™ reagents can be used for staining antigen-specific T cells and flow cytometric analysis. In humans, the MHC molecules are called HLA (Human Leukocyte Antigen).</div></div>

[STEPS]
?. [Peptide Exchange:]
Bring all reagents to 0°C by putting them on ice.
?. [Peptide Exchange:]
Dilute 10mM stock solutions of peptides of choice to 400µM by mixing 5µl of peptide stock solution with 120µl PBS, and keep on ice.
?. [Peptide Exchange:]
Add 20µl diluted peptide (400µM) and 20µl peptide Flex-T™ monomer UVX (200µg/ml) into 96-well V bottom plate. Mix by pipetting up and down.
?. [Peptide Exchange:]
Seal the plate; centrifuge at 2500xg for 2 minutes at 4°C to collect the liquid down
?. [Peptide Exchange:]
Remove the seal; put the plate on ice and illuminate with UV light for 30 minutes (the distance of the UV lamp to the samples should be 2-5cm).
?. [Peptide Exchange:]
Seal the plate; incubate for 30 minutes at 37°C in the dark.
?. [Peptide Exchange:]
To evaluate the efficiency of the peptide exchange follow the Protocol for HLA class I ELISA to evaluate peptide exchange.
?. [Generation of Tetramers:]
Transfer 30µl of peptide-exchanged monomer into a 1.5ml Eppendorf tube, or a new plate, then add 3.3µl of conjugated streptavidin, mix by pipetting up-and-down. Incubate on ice in the dark for 30 minutes. This is enough for about 15 tests.Note: BioLegend fluorophore-conjugated streptavidin products are recommended. For 30µl of exchanged Flex-T™ monomer we suggest using 3.3µl of BioLegend PE-streptavidin (Cat#405203) or APC- streptavidin (Cat#405207). For BV421-streptavidin conjugate (Cat#405225) use 1.3 µl. For oligo barcoded Streptavidin reagents please use 1.3 µl. For optimal reaction with other fluorophore-conjugated streptavidin products ensure that the monomer:streptavidin conjugate has a 5:1 ~ 6:1 molar ratio.(For our full choice of Streptavidin conjugates, visit: www.biolegend.com/streptavidin_conjugates. Note that purified, biotinylated, HRP, MojoSort™, and Ultra Streptavidin (USA) kits are not recommended for this procedure.)
?. [Generation of Tetramers:]
During the incubation, prepare blocking solution by adding 1.6µl 50mM D-Biotin and 6µl 10% (w/v) NaN3 to 192.4µl PBS, mix by vortexing. After the incubation, add 2.4µl of blocking solution and pipette up-and-down to stop the reaction.
?. [Generation of Tetramers:]
Incubate the tubes or sealed plates at 2-8°C overnight (or on ice for 30 minutes in the dark, if staining needs to be performed immediately).Tip: We recommend Flex-T™ to be assembled with two different streptavidin conjugates in separate reactions. This allows for two-color staining with the same tetramer allele, ensuring the highest specificity.
?. [Cell Staining and Flow Cytometric Analysis:]
Prepare cells of interest
?. [Cell Staining and Flow Cytometric Analysis:]
Prior to performing staining, centrifuge the assembled tetramers in tube or plate at 2500xg for 5 minutes at 4°C. Then keep on ice in the dark.
?. [Cell Staining and Flow Cytometric Analysis:]
Add 2 x 106 cells to 12 x 75mm tubes or a 96-well U-bottom plate. Adjust volume to 200µl with Cell Staining Buffer. Add 2µl per sample of Flex-T™ complex prepared in Steps 7-9, mix and incubate on ice in the dark for 30 minutes.
?. [Cell Staining and Flow Cytometric Analysis:]
If co-staining with surface antibodies, prepare the antibody cocktail based on optimal staining concentration of each reagent. Incubate for 30 minutes on ice in the dark.
?. [Cell Staining and Flow Cytometric Analysis:]
Wash the cells with Staining Buffer two times. Resuspend cells with Staining Buffer.
?. [Cell Staining and Flow Cytometric Analysis:]
Acquire the samples with a flow cytometer and appropriate settings within 2 hours.Tip: A titration of the Flex-T™ is recommended for optimal performance.