Transcription of 流式细胞术的原理与应用 - CAS
1 1 FACS: fluorescence-activated cell sorting 2 FACS Aria 3 4 5 10 / 99% 6 BD FACS Aria 7 8 488nm ,633nm 405nm 375nm 561nm 9 407nm 633nm 488nm 10 460 500 540 460 500 540 460 500 540 SP 500 LP 500 BP 500/50 Longpass(LP) Shortpass(SP) Bandpass(BP) 11 PMT 12 FACS Aria BP LP 13 BD FACS Aria 14 PBS 15 16 1.
2 2. 3. 17 5ml 15mL ACDU 6 8 24 96 384 18 1~10 sort confocal 19 BD FACS Aria 20 21 beads 106 1 107 /ml 1ml 300 22 + 23 23 + - 1 + 2 + 1 + 2 24 (L1) FITC Alexa Fluro 488 488nm PE PE-Texas Red PE-Cy5 PerCP PE-Cy7 (L2) APC Alexa Fluro 647 633nm APC-Cy7 (L3) Alexa Fluro 405 Pacific Blue 405nm PI 488nm 520nm FITC AmCyan BD Horizon V500 25 ( FITC GFP CFSE (PMT) PE FITC PerCP PE FITC 26 FreqFluorescence FALS Sensor Fluorescence detector (PMT3, PMT4 etc.))
3 FSC SSC + 27 FSC Forward Scatter SSC Side Scatter 28 (FSC) FSC 29 SSC) SSC : 30 31 FSC SSC 32 Histogram (Dot Plot) (Contour Plot) (Density Plot) (3D Plot) 33 Histogram ( ) 34 (Dot Plot) 35 Contour Plot Density Plot 36 37 DNA ROS) (FRET)
4 38 DNA 39 DNA - G0/G1 2n S 2n~4n G2/M 4n 40 DNA - PI DNA Modfit G0/G1 G2/M S DNA ES 41 DNA 70% RNase RNA PI 37 C 10min DNA modfit 42 DNA FSC-SSC FL2-W 43 DNA DNA DNA G0/G1 sub G1 PI G0/G1 S G2/M S G2/M 44 2n 4n 45 DNA DNA DNA DNA PI) DNA FITC 46 47 DNA 48 - annexin V/PI PS PS annexin -V Ca2+ PS annexin -V PI: PI DNA PI PI DNA PS FITC annexin -V PS 49 annexinV-/PI- annexinV+/PI- annexinV+/PI+ CB12 293T FL1:FITC-annexinV, FL2.
5 PI annexin V/PI 50 -Rhodamine123 Rhodamine123 Rhodamine123 Mean Mean 51 caspase-3 caspase-3 caspase-3 - caspase-3 52 TUNEL 1. DNA DNA 3 OH 3 OH DNA 2. TdT DNA I Brdu Brdu DNA 3 OH 3. FITC Brdu 53 54 CFSE VPD450 PKH DNA Brdu 55 CFSE) PKH26,PKH-67) 56 CFSE CFDA-SE CFSE CFSE CFSE 2 57 - VPD450 VPD450 CFSE FL1 CFSE) 407nm 450nm 58 Brdu Brdu DNA.
6 2003 PMA 8h T Brdu Brdu 59 60 PKH: PKH26 FL2, PKH67 FL1 61 ROS) 62 ROS -DCFH-DA 2 ,7 - DCFH-DA) ROS DCFH, DCFH H2O2,O2- ROS DCF DCF:488nm 525nm DCF DCF DCFH-DA DCFH DCF DCF: FL1) PI: FL2 63 64 CBA cytometric bead array): 65 1 2 BFA( 3 4 5 66 CBA(cytometric bead array) ) PE 67 CBA(cytometric bead array) Y FL3 PE-CY5 6 X FL2 PE 68 CBA(cytometric bead array) FSC/SSC FL3 (PE-cy5)
7 6 6 R1 69 70 CBA Flex Set ABCDEFGHI123456789FL4-H or Red-AFL3-H or NIR-AABCDEFGHI123456789FL4-H or Red-AFL3-H or NIR-A Flex Set FL3,FL4 FL2-PE 71 72 GFP GFP RNA GFP RNA 73 74 FRET) B ECFP EYFP No Binding: 407nm 475nm A A B ECFP EYFP Binding: 407nm 530nm FRET 1 2 3-6nm FRET 50% 75 FRET) A B B ECFP EYFP No Binding: 407nm 475nm A A B ECFP EYFP Binding: 407nm 530nm A B ECFP EYPF FRET 407nm FRET 100% 530nm 475nm A B ECFP EYPF FRET 407nm 475nm 76 FRET 77 FRET YPetD CyPet FACSAria FRET FACSAria FRET KD,DNA Xia you,et protein interaction mapping with FRET ,18458-184631(2006) 78
