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| MATERIALS AND METHODS Chemicals, Cell Lines, Cell Culture, and Immunological Reagents All common laboratory chemicals, proteinase inhibitors, phorbol-12 myristate 13-acetate PMA ; , trifluoperazine TFP ; , N- 6-aminohexyl ; W7 ; , calmidazolium CLM ; , PD98059, wortman, LY290042, prevanadate, and anti-FLAG-M2 monoclonal antibody mAb ; and its agarose conjugates were purchased from Sigma St. Louis, MO ; . The CMK based furin inhibitor, decRVKR-CMK ; , and a matrix metalloproteinase inhibitor, ilomastat GM6001 ; , were purchased from BACHEM Philadephia, PA ; . TIMP-3 was purchased from R & D Systems Minneapolis, MN ; . Restriction enzymes were purchased from Promega Madison, WI ; or Invitrogen Gaithersburg, MD ; . COS1 and Madin Darby canine kidney MDCK ; cells and its derivatives were maintained as described 45 ; . Dulbecco's modified Eagle's medium DMEM ; was purchased from Invitrogen Gibco BRL Gaithersburg, MD ; . Fetal bovine serum FBS ; , penicillin G, and streptomycin were purchased from Life Technologies Rockville, MD ; . 2-M was purchased from Roche Molecular Biochemicals Indianapolis, IN ; . Rabbit polyclonal hADAM19 antibodies pAb361 antimetalloproteinase domain, anti-Cat ; and pAb362 anti-disintegrin domain, anti-Dis ; were generated by our laboratory as reported 50 and trimethoprim.
Figure 3. Purification of TCR + DN T cells via magnetic beads and flow cytometric sorting and TCR V repertoire of purified DN T cells. A ; Purification of TCR + DN T cells using a Dynabeads-based depletion system to eliminate CD4 + , CD8 + , CD14 + , CD19 + , and CD56 + cells, as well as flow cytometric sorting with anti-TCR, anti-CD4, and anti-CD8 mAbs. B ; Comparative analysis of the TCR V repertoire of purified DN and CD4 + T cells. C ; Total RNA from sorted DN T cells was extracted, reverse transcribed, and amplified by RTPCR using V primers. Amplified cDNA was copied with a fluorescent C primer in a runoff reaction and subjected to electrophoresis on an automated sequencer. The patterns obtained show the size in amino acids of the CDR3 region x-axis ; and the relative fluorescence intensity y-axis ; of in-frame V-C amplification products.
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