TITLE: Flex-T™ Fixed Peptide Tetramer Preparation and Flow Cytometry Staining Protocol
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is optimized to generate MHC tetramers using our streptavidin fluorophore conjugates and fixed peptide monomers. Unlike our UV-exchangeable monomers (denoted as UVX), the fixed peptide monomers are ready to use and do not need a peptide exchange. The resulting Flex-T™ tetramers 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]
?. [Generation of Tetramers:]
During the incubation, prepare blocking solution by adding 1.6 µL 50 mM D-Biotin and 6 µL 10% (w/v) NaN3to 192.4 µL PBS and mix by vortexing. After the incubation, add 2.4 µL of blocking solution and pipette up and down to stop the reaction.
?. [Generation of Tetramers:]
Bring all reagents to 0°C by putting them on ice.
?. [Generation of Tetramers:]
Transfer 30 µL of fixed peptide monomer into a 1.5 mL Eppendorf tube, or a new plate, then add 3.3 µL of conjugated streptavidin and 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 30 µL of fixed peptide Flex-T™ monomer, we suggest using 3.3 µL of BioLegend PE Streptavidin (Cat. No. 405203) or APC Streptavidin (Cat. No. 405207). For BV421™ Streptavidin (Cat. No. 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 our Streptavidin Conjugates webpage. Note that purified, biotinylated, HRP-Streptavidin and MojoSort™ and Ultra Streptavidin (USA) kits are not recommended for this procedure.)
?. [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.
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?. [Cell Staining and Flow Cytometric Analysis:]
Prepare cells of interest.
?. 1.Prepare cells of interest
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?. 2.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.
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?. 3.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
?. minutes.
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?. 4.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.
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?. 5.Wash the cells with Staining Buffer two times. Resuspend cells with Staining Buffer.
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?. 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.
?. [Cell Staining and Flow Cytometric Analysis:]
Prior to performing staining, centrifuge the assembled tetramers in tubes or a plate at 2500 xgfor 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 75 mm tubes or a 96-well U-bottom plate. Adjust volume to 200 µL with Cell Staining Buffer. Add 2 µL 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 on a flow cytometer with appropriate settings within 2 hours.Tip: A titration of the Flex-T™ is recommended for optimal performance.