Product name | Code No. | ||
---|---|---|---|
PE-labeled | APC-labeled | BV421-labeled | |
QuickSwitch™ Quant HLA-A*01:01 Tetramer Kit | TB-7308-K1 | TB-7308-K2 | TB-7308-K4 |
QuickSwitch™ Quant HLA-A*02:01 Tetramer Kit | TB-7300-K1 | TB-7300-K2 | ― |
QuickSwitch™ Quant HLA-A*03:01 Tetramer Kit | TB-7306-K1 | TB-7306-K2 | TB-7306-K4 |
QuickSwitch™ Quant HLA-A*11:01 Tetramer Kit | TB-7304-K1 | TB-7304-K2 | TB-7304-K4 |
QuickSwitch™ Quant HLA-A*24:02 Tetramer Kit | TB-7302-K1 | ― | TB-7302-K4 |
QuickSwitch™ Quant H-2Kb Tetramer Kit | TB-7400-K1 | TB-7400-K2 | TB-7400-K4 |
Component name | Description | Size | Storage | |
---|---|---|---|---|
QuickSwitch™ Tetramer | Exiting Peptide loaded MHC tetramer | 500 µL | 2-8°C | |
Peptide Exchange Factor | The proprietary peptide exchange factor | 13 µL | -20°C | |
Magnetic Capture Beads | Magnetic beads conjugated with a capture antibody specific for tetramers | 500 µL | 2-8°C | |
Exiting Peptide Antibody-FITC (25x) | FITC conjugated antibody reacting against Exiting Peptide | 25 µL | 2-8°C | |
Reference Peptide 1 mM | The peptide for a positive control | 13 µL | -20°C | |
Assay Buffer (10x) | Washing/Dissolving buffer | 1.7 mL | 2-8°C |
■ Peptides for new specificity tetramers | ■ Flow cytometer |
■ Magnetic tray for microplate | ■ Vortex |
■ Microtubes | ■ Round or conical bottom microplates |
■ Micropippettes and disposable tips | ■ Aluminum foil |
■ Ultra pure water | ■ DMSO |
■ Plate shaker (Labline model 4625 or equivalent) | |
■ Sonicator (Branson Ultrasonic Cleaner Model #B200 or equivalent) |
WARNINGS AND PRECAUTIONS
1 Dissolve each lyophilized peptide to be assayed in DMSO to a 10 mM solution.
(For high affinity peptides, a 1 mM stock solution is a reasonable starting concentration for the assay.)
2 Pipet 50 µL of QuickSwitch™ Tetramer into a microtube.
3 Add 1 µL of peptide and mix gently with pipetting.
4 Add 1 µL of Peptide Exchange Factor and mix gently with pipetting.
5 Repeat steps 1 - 4 for each additional peptide.
6 Prepare Reference Peptide as follows;
(1) Pipet 50 µL of QuickSwitch™ Tetramer into a microtube.
(2) Add 1 µL of Reference Peptide (1 mM) and mix gently with pipetting.
(3) Add 1 µL of Peptide Exchange Factor and mix gently with pipetting.
7 Incubate at least for 4 hours* at room temperature protected from light.
(*Reaction time varies depending on the kit. For details, please refer to the datasheet for each kit.)
8 Refrigerate tetramers at 2-8°C protected from light when not used.
The following assay measures the percentage of original peptide replaced by a competing peptide to help determine whether the resulting tetramer is suitable for antigen-specific CD8+ T cell staining. All control samples are required for the assay. The QuickSwitch™ Calculator on the website can be downloaded for determining percentages of peptide exchange.
WEB
http://www.mblintl.com/quickswitch-peptide-exchange-calculator/
Control #3: Beads that have captured QuickSwitch™ Tetramer. FITC conjugated antibody reacts against Exiting Peptide.
Reference Peptide: This peptide typically undergoes about 100% exchange. FITC conjugated antibody does not react against Reference Peptide.
User’s Peptide: Peptides selected by users. The figure above shows the flow cytometry data for 50% peptide exchange.
1 Prepare 1x Assay Buffer as follows:
For 1-5 peptide exchanges, prepare 7.5 mL by mixing 750 µL of 10x concentrated Assay Buffer with 6.75 mL of distilled water.
For 6-8 exchanges, double the volumes.
2 Immediately before use, vortex the tetramer capture beads for 30 seconds, followed by a 30-second sonication in a water bath sonicator. If no sonicator is available, vortex an additional 30 seconds.
3 Prepare a round or conical-bottom 96 well microtiter plate.
4 Pipet 20 µL Magnetic Capture Beads into each of wells #1-#7. Pipet the beads into wells for three essential
controls, Reference Peptide, and tetramers generated in Step A. The below figure shows the case when three new tetramers are used.
5 Pipet 5 µL 1x Assay Buffer in well #2.
6 Pipet 5 µL QuickSwitch™ Tetramer in wells #1 and #3.
7 In wells #4-#7, pipet 5 µL taken from the difference peptide exchange microtubes as described in Step A.
8 Shake plate for 45 min. at 550 rpm, protected from light with an opaque cover such as a piece of aluminum foil.
9 Dispense 150 µL of 1x Assay Buffer in wells #1-#7.
10 Place the plate on a plate magnet for 5 min. to sediment beads.※
11 Flick the plate held tightly to the magnet and blot on a paper towel to minimize cross-contamination of wells.
12 After returning the plate upright, vortex for 2 seconds to disperse the beads.
※It is possible to pellet by centrifugation using a plate holder at 1,000 x g for 5 min.
13 Dilute 25x Exiting Peptide Antibody to 1x as follows: Determine the number (n) of samples to stain with the antibody, including control #2, control #3, Reference Peptides and tetramers generated in Step A. Add one (+1), to account for pipetting errors. In a microtube, pipet (n+1) x 24 µL of Assay Buffer and then add (n+1) x 1 µL of Exiting Peptide Antibody-FITC. Mix by pipetting.
14 Pipet 25 μL of 1x Exiting Peptide Antibody-FITC prepared in step 13 in wells #2-#7.
15 Pipet 25 µL of 1x Assay Buffer in well #1.
16 Shake plate for 45 min. at 550 rpm, protected from light.
17 Dispense 150 µL of 1x Assay Buffer in wells #1-#7.
18 Place the plate on a plate magnet for 5 min. to sediment beads.※
19 Flick the plate held tightly to the magnet and blot on a paper towel to minimize cross-contamination of wells.
20 After returning the plate upright, vortex for 2 seconds to disperse the beads. Then remove the plate from the magnet.
21 Resuspend beads in 200 µL 1x Assay Buffer.
※It is possible to pellet by centrifugation using a plate holder at 1,000 x g for 5 min.
1 Pipet 200 µL 1x Assay Buffer and 5 µL Magnetic Capture Beads into well #X.
Now they are ready to be analyzed by flow cytometer.
For more detailed information, please see the product datasheet.
The QuickSwitch™ Calculator can be downloaded for determining percentages of peptide exchange.
Please visit the website at https://www.mblintl.com/quickswitch-peptide-exchange-calculator/.
Tetramers bind to T cell receptors (TCR) via three MHC/peptide monomers. Therefore the minimal recommended peptide exchange percentage should be 75%.