MACS magnetic beads based cell purification FITC conjugated antibody Add anti-FITC microbeads CD8＋Cells MNC anti-FITC microbeads CD8ーcells only CD8＋ cells are captured FITC conjugated anti-CD8 antibody CD8＋cells are collected magnetic field flow through: CD8ーcells CD8＋cells MACS-beads-based sorting of CD8+ cells Cell type No. of calculated R1gate cells/total M1 region cells/ M1 region cells/ cells cells (%) R1 gate cells (%) total cells (%) MNC 4.5x107 CD8(-) cells 3.3x107 43.3 CD8(+) cells 1.4x107 94.4 46.2 estimated% recovery 15.8 7.10×106 100.0 14.3 6.2 2.04×106 28.7 99.6 94.0 1.32×107 34.2 001 EO MNC.001 Estimated No. of T cells 002 EO CD8(-).002 185.1 003 EO CD8(+).003 EO 46.2% 0 200 400 600 800 1000 FSC 43.3% 0 001 EO MNC.001 100 200 400 600 800 1000 FSC CD8+cells/R1 34.2% 14.3% 102 103 FITC-CD8 0 002 EO CD8(-).002 CD8+cells/R1 101 94.4% 104 100 101 102 103 FITC-CD8 200 400 600 800 1000 FSC 003 EO CD8(+).003 CD8+cells/R1 99.6% 104 100 101 102 103 FITC-CD8 104 140416 Induction plan of antigen specific CTLs by PMDC11 Responder Stimulator peptide CD8+ T cells (EO) 1.0×107/well PMDC11-3 1.0×106/well mWT1 10µg/ml Medium：5%AS RPMI1640 medium( 6 well plate) Cytokine：IL-2(50 U/ml), IL-7(10 ng/ml) 140416 PMDC刺激培養法 mWT1ペプチド 10µg/ml 末梢血採取 5U/mlトロンビン加 37℃,1〜2時間 血漿 血清 PMDC 細胞数 1×106/ml 3,000rpm 10分間遠心 末梢血単核球 24hr, 37℃ 6wellプレート irradiation 30Gy 5% 自己血清加培養液 (RPMI1640) 細胞：1.0×107個/well 6well プレートに播種 PMDCによる抗原提示 mWT1ペプチド 細胞数：1.0×106/well IL-2 50U/ml，IL-7 10ng/ml加培地で培養 TCR CD8＋T細胞 培養1週後 活性化 mWT1ペプチド 特異的T細胞受容体 mWT1ペプチド特異的CTL mWT1ペプチド特異的CTLの割合をFACSを用いて解析する。 ＊刺激、解析は毎週行う
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