TCRExpressTM Mouse TCR V ββββ Repertoire CDR3 Diversity Determination (Spectratyping) and

Quantitative Analysis Kit

Cat. No. M0524 Size: 2 sets (22 V ββββ families/each, with enzymes)

M0525 Size: 4 sets (22 V ββββ families/each, with enzymes)

M0526 Size: 8 sets (22 V ββββ families/each, with enzymes)

Upon receipt of kit, take out enzymes and store at -20°C. Store balance of kit at 4°C.

Description The TCRExpressTM Mouse T Cell Receptor (TCR) Vββββ Repertoire CDR3 Diversity Determination (Spectratyping) and Quantitative Analysis System is designed for the convenient, sensitive, reproducible, and quantitative determination of changes in CDR3 diversity (clonality) of the TCR Vβ repertoires in mouse T lymphocytes. The system consists of three major components: primary and then nested PCR amplification, CDR3 diversity determination, and quantitative analysis of the CDR3 diversity with proprietary software. The uniquely designed primers and essential reagents have been pre-loaded in a 24-well PCR plate with chimney wells, which is more effective than flat-top plates in preventing contamination. For high resolution spectratyping and easy to perform molecular cloning, seven non-template nucleotides are added to the 5’end of the primers and all of the Vβ primers are labeled with 6-FAM fluorescence. Proprietary PCR reaction buffers and conditions play a critical role, resulting in highly sensitive and specific PCR amplifications. The analysis system is a DNA fragment - based assay. The amplified nested PCR products labeled with 6-FAM can be analyzed for size on many kinds of DNA sequencers using the manufacturer’s fragment sizing software. For statistical and quantitative analysis of the CDR3 diversity, our system provides powerful proprietary software. There are two panels (control and testing panel) in the software. The Control panel is used to establish a standard distribution for each PCR CDR3 length of each Vβ family. The extent of changes of CDR3 diversity is defined as D (distance from mean value). D > 3 SD (standard deviations) of each fragment length of each family indicates significant changes in the Vβ family (see the software instruction). The system can detect 22 individual Vβ gene families (from Vβ1 to 20, with Vβ 8 having three subfamilies, Vβ 8.1, Vβ 8.2 and Vβ 8.3) in Mouse T lymphocytes and their subsets, such as CD4, CD8 and CD45RA and CD45RO T cell subsets of the CD4 and CD8 populations. The kit can be used to determine quantitatively changes in CDR3 diversity (clonality) which are induced by viral, tumor, or self-immune antigen response and document the efficacy of vaccines or immunologic therapy in mouse model. Users need to prepare cDNA from T cells before using this kit.

Contents

Purpose Name M0524 M0525 M0526 MQ plate I* 2 4 8

MQ plate II** 2 4 8 PCR reaction buffer I*** 200 µl 400 µl 800 µl PCR reaction buffer II*** 200 µl 400 µl 800 µl Molecular grade water 4 ml 8 ml 16 ml

For PCR amplification

Taq DNA polymerase 24 µl 48 µl 96 µl For CDR3

diversity analysis Proprietary software 1 1 1

* MQ plate I is designed to perform primary PCR amplification of 22 Vβ gene families (Vβ1 to Vβ20, thereof Vβ8 with three sub families, Vβ 8.1, Vβ 8.2 and Vβ 8.3). ** In MQ plate II, the 22 nested Vβ primers are labeled with 6-FAM.

Protocol cDNA (prepared by user)

↓ PCR amplifications

↓ CDR3 diversity determination (spectratyping)

↓ CDR3 diversity analysis using proprietary software

A) PCR amplifications 1. Primary PCR amplification in MQ plate I with 24 tubes

•••• Open the foil-based self sealing bag labeled MQ plate I (note the plate and the plate user manual card) For sample tubes (22 Vββββ families) While keeping the MQ plate I on ice, add the following to each well or make a master mix of the following reagents for each family desired, and aliquot x µl into each sample well of the plate: cDNA x µl PCR reaction buffer I 2.5 µl Taq DNA polymerase (5U/µl) 0.25 µl Add molecular grade water to receive total final volume of 25.0 µl

For control tubes (positive and negative) Add the following reagents to the appropriate tubes: PCR reaction buffer I 2.5 µl Taq DNA polymerase (5U/µl) 0.25 µl Molecular grade water 22.25 µl

Cover the wells with the provided strip caps and gently vortex the plate to mix. Centrifuge briefly to collect all the reagents at the bottom of the wells or gently tap the plate on a solid surface. Perform PCR amplification in a thermocycler using the following parameters: PCR amplification conditions:

1) Initial denaturation at 95oC for 3 min, then

2) 95oC for 30 sec 3) 55oC for 30 sec 4) 72oC for 45 sec Repeat steps 2 to 4, for 35 cycles

5) 72oC for 5 min Store PCR products at -20 oC.

2. Nested PCR amplification in MQ plate II with 24 wells •••• Open the foil-based self sealing bag labeled MQ plate II (note the plate and the plate user

manual card) While keeping the MQ plate II on ice, add the following to each well or make a master mix of the following reagents for each family desired plus the controls, and aliquot x µl into each sample and control well of the plate: PCR reaction buffer II 2.5 µl Taq DNA polymerase (5U/µl) 0.25 µl Molecular grade water 21.25 µl

Transfer carefully 1 µl of the primary PCR Products with multichannel pipette from each sample and control well in MQ plate I to the corresponding wells (samples and controls) in the MQ plate II. Cover the wells with the provided strip caps and gently vortex the plate to mix. Centrifuge briefly to collect all the reagents at the bottom of the wells or gently tap the plate on a solid surface. Perform PCR amplification in a thermocycler using the following parameters: PCR amplification conditions:

1) Initial denaturation at 95oC for 3 min, then

2) 95oC for 30 sec 3) 55oC for 30 sec 4) 72oC for 30 sec Repeat steps 2 to 4, for 25 cycles

5) 72oC for 10 min Store PCR products at -20 oC.

B) CDR3 size determination (spectratyping)

1. Checking presence of PCR products Mix 3 µl of the nested PCR product with 0.5 µl of 6 x DNA loading dye, then electrophorese in a 2% agarose (molecular biology grade, not provided by kit) gel. Successful PCR amplifications should display a defined DNA band on the agarose gel from positive control and each sample (each Vβ family) and no PCR product from the negative control well. PCR product sizes of 22 Vβ gene families should be between 120 to 240 bp.

2. TCR CDR3 size determination (spectratyping) Samples for analysis on a DNA sequencer are prepared based on the manufacturer’s suggestions and

using Rox 400 as the size standard. The size of the individual PCR products will be determined by the sequencer’s software and should be output as an Excel file for use in the kit’s proprietary software.

C) CDR3 diversity analysis Quantitative determination of CDR3 diversity of 22 Vβ families can be performed using the proprietary statistical quantitative software package provided in the kit. There are two panels (control and testing panel) in the software. The Control panel is used to establish a standard distribution for size range of each Vβ family. An average distribution of each CDR3 length within the determined T cell population is based on six to ten control samples, which are subjected to the same PCR amplification and spectratyping conditions as the final test samples. The extent of the change in the CDR3 diversity is defined as D (distance from mean value). D > 3 SD (standard deviation) in the fragment length of each family indicates that there are significant changes in the Vβ family (see the software instruction).

Optional : CDR3 region sequencing

A TA cloning kit (not supplied with the kit) is recommended.

Purify 20 µl of PCR products from the Vβ family PCR of interest using a PCR purification kit. Following the manufacturers’ directions, ligate and transform the DNA fragment into the cloning vector and cells for subsequent sequencing. The M13 forward or reverse primer is recommended for sequencing.

Troubleshooting guide Problem Likely Cause Suggestions No signal in samples and positive control

Sample and control cDNA were not amplified

Be sure the Taq polymerase and PCR reaction buffer were added

No signal in very few Vβ families but signal appears in most Vβ families and the positive control

cDNA concentration is too low or there are deletions of the individual Vβ family from progressing cells

Increase cDNA concentration by increasing the starting number of T cells

Signal appears in negative control well

Contamination during PCR preparation

Decontaminate all equipment, including pipettes. Use filtered pipette tips.

Poor resolution of CDR3 patterns from in gel visualization

PCR product concentration is too high or too low

Adjust ratio of PCR products with distilled water before running on gel