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 .
