Flex-T™ Tetramer and Cell Staining Protocol
Bring all reagents to 0°C by putting them on ice.
Dilute 10mM stock solutions of peptides of choice to 400μM by mixing 5μl of peptide stock solution with 120μl PBS, and keep on ice.
Add 20μl diluted peptide (400μM) and 20μl conditional Flex-T™ monomer (200μg/mL) into 96-well U-bottom plate.
Mix by pipetting up and down.
Seal the plate; centrifuge at 3300xg for 2 minutes at 4°C to collect the liquid down.
Remove the seal; put the plate on ice and illuminate with UV light for 30 minutes (the distance of the UVlamp to the samples should be 2-5 cm).
Seal the plate; incubate for 30 minutes at 37°C in the dark.
To evaluate the efficiency of the peptide exchange follow the Protocol for HLA class I ELISA to evaluatepeptide exchange.
Transfer 30μl of peptide-exchanged monomer into 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 enoughfor about 15 tests.
Note: BioLegend fluorophore-conjugated streptavidin products are recommended.
For 30μl ofexchanged Flex-T™ monomer we suggest to use 3.3μl of BioLegend PE-streptavidin (Cat#405203) or APC- streptavidin (Cat#405207).
For BV421-streptavidin conjugate (Cat#405225) use 1.3μl.
For optimal reaction with other fluorophore-conjugated streptavidin products ensure that themonomer:streptavidin conjugate has a 6:1 molar ratio.
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.
Seal the plate and incubate at 2 - 8°C overnight (or on ice for 30 minutes in the dark).
Note: We recommend Flex-T™ to be assembled with two different streptavidin conjugates inseparate reactions.
This allows for two-color staining with the same tetramer allele, ensuring thehighest specificity.
Prepare cells of interest. 
Prior to perform staining, centrifuge the plate at 3300xg for 5 minutes at 4°C.
Keep on ice in the dark.
Add 2 x 106 cells to a 96-well U-bottom plate or 12 x 75 mm tubes.
Adjust volume to 200μl with CellStaining Buffer.
Add 2μl per sample of Flex-T™ complex prepared in Steps 7 - 9, mix and incubate on icein the dark for 30 minutes.
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.
Wash the cells with Staining Buffer two times.
Resuspend cells with Staining Buffer.
Acquire the samples with a flow cytometer and appropriate settings within 2 hours.
Note: A titration of the Flex-T™ is recommended for optimal performance.
