Flex-T™ Fixed Peptide Tetramer Preparation and Flow Cytometry Staining Protocol

 

Background

 

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).

 

Reagents

• Phosphate buffered saline pH 7.4, 10X concentrate (PBS, BioLegend Cat. No. 926201)
• Fixed peptide Flex-T™ biotin monomer
• 50 mM D-Biotin (e.g. Thermo Fisher, Cat. No. B20656)
• 10% (w/v) NaN₃ (e.g. Sigma, Cat. No. S2002)
• Fluorophore-conjugated Streptavidin (BioLegend Cat. No. 405203, 405207, 405225 or equivalent)
• Cell Staining Buffer (BioLegend Cat. No. 420201 or equivalent)
• 1.5 mL tubes (e.g. Eppendorf Cat. No. 022364111)
• For proteogenomic applications compatible with our TotalSeq™ product line, use one of our oligo barcoded fluorophore-conjugated Streptavidin reagents.

 

Equipment

• Centrifuge capable of accommodating microtiter plates and tubes
• Single and multichannel pipettes capable of accurate delivery of variable volumes 
• Pipette tips

 

Tips

• Avoid repeated freeze-thawing.
• Avoid exposure to light as much as possible when performing this protocol. Do not work in front of a window.
• Centrifuge all vials before use (1 minute 2500 x g at 4°C).

 

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Generation of Tetramers

1. Bring all reagents to 0°C by putting them on ice.
2. 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.)
3. During the incubation, prepare blocking solution by adding 1.6 µL 50 mM D-Biotin and 6 µL 10% (w/v) NaN₃ to 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. 
4. 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

5. Prepare cells of interest.
6. Prior to performing staining, centrifuge the assembled tetramers in tubes or a plate at 2500 x g for 5 minutes at 4°C. Then keep on ice in the dark.
7. 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.
8. 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.
9. Wash the cells with Staining Buffer two times. Resuspend cells with Staining Buffer.  
10. 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.