Flow Cytometry Workshop:

Transcription

1 Flow Cytometry Workshop: Section III: DNA Content Analysis Beckman Coulter Taiwan Branch 技 術 支 援 經 理 楊 人 泰

2 DNA Content Analysis-common sense DNA content analysis is one of the oldest Flow applications. Using appropriate dye to label DNA and then measuring the fluorescence by flow cytometry DNA dyes using on flow cytometry: - Propidium Iodide (PI) - 7-AAD - Acridine Orange (AO) - DAPI Familiar applications using this technology: - Cell cycle analysis - Ploidy analysis - DNA index analysis - Apoptosis sub-g1 analysis - Hoechst 33342, Vybrant DyeCycle

3 Application 1: cell cycle analysis Cells contain different DNA content through cell cycle 4C 4C G2 M 2C 2C~4C S 2C G1 G0 Quiescent cells

4 Application 1: cell cycle analysis

5 Application 1: cell cycle analysis 4C G2 4C M 2C Stained with PI, analyzed by Flow 2C~4C S 2C G1 G0 Cell Numbers 2C 2C~4C 4C DNA content or FL intensity

6 Application 1: cell cycle analysis G 0 -G 1 % G 2 -M % # of Events S % Fluorescence Intensity

7 Application 1: cell cycle analysis Cell cycle (PI stained) + mataphase specific protein (phospho-histon-h3)

8 Application 1: cell cycle analysis BromodeoxyUridine (BrdU) Incorporation Control (left) versus drug treated (right) effect on cell cycle

9 Application 2: ploidy analysis Hyperdiploid: greater than the normal 2n number of chromosomes Hypodiploid: Less than the normal 2n number of chromosomes Tetraploidy: Containing double the number of chromosomes

10 Application 3: DNA index analysis

11 Application 4: apoptosis (sub-g0/g1) analysis DNA Content Measurement : Sub-G0/G1 Control Drug treated

12 The process: cell stained with PI, gating the major population from SS/FS plot and display gated cell on fluorescent (FL2 or FL3) histogram But PI usually bring the doublet problem..? Doublet Problem SS FS FL2 or FL3 How to gate out the doublet cell before FL plot display??

13 The signaling process by Flow A B C There are 3 kinds of signals could be measured (H) (A) Time of Flight (W) In FACSan and FACSCalibur, we used to used H as the major parameter In FC500, XL, and FACSCanto, we used to used A as the major parameter

14 To measure the signals among singlets and doublet : Peak (H) Integral (A) TOF (W) 1 1 2

15 Doublet discrimination: method 1, using H and A Peak (H) Integral (A)

16 Doublet discrimination: method 1, using H and A You could use this method on XL, FC500, FACSCanto, Cyan.

17 Doublet discrimination: method 2, using H, A and H/A Peak (H) Integral (A) Ratio (H/A) 1 1 ½ or <1

18 Doublet discrimination: method 2, using H, A and H/A You could use this method on XL, FC500..

19 Doublet discrimination: method 3, using A and W Integral (A) Tim of Flight (W)

20 Doublet discrimination: method 3, using A and W You could use this method on FACScan, FACSCalibur, FACSCanto.

21 So the real process are. SS FS FL2 or FL3

22 Cell cycle analysis: real practice on FC Parameters selection

23 2. Plots Creation 3. Instrument Setup: Run Control Cell Staining with PI: 調 整 FS SS FL3 Lin(Int) FL3 Peak Voltage & Gain

24 Final Result

25 Other critical issues: 1. The proficiency of sample preparation: the CV of G0G1 phase Good resolution Bad resolution Solution:

26 Other critical issues: 2. How to resolve GoG1 / S / G2M phase? Manually G 0 -G 1 % G 2 -M % By Software: Phoenix MultiCycle or Verity Mofit # of Events S % Fluorescence Intensity

27 Some Flow relative web-sites: Purdue University Cytometry Laboratories Purdue University Cytometry Laboratories Archive 陽 明 大 學 儀 器 中 心 及 Flow 討 論 區 NCI ETI Branch Flow Cytometry Core Laboratory Invitrogen s Fluorescence Spectra Viewer