流式細胞儀在 免疫細胞生物學上之應用

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(1)

流式細胞儀在

免疫細胞生物學上之應用

謝長奇

(2)

流式細胞儀應用

A brief list of applications that use flow cytometers includes:

Disease diagnosis

Chromosome karyotyping

Cell function analysis

Cancer therapy monitoring

Detecting fetal cells

Cell kinetics

Identifying tumor cells

Cytogenetics

Fundamental cell biology

(3)

流式細胞儀應用

免疫細胞分析

Cell surface marker analysis

Intracellular cytokines analysis

Platelet analysis

Insulin resistance

細胞凋亡

上樣與資料分析

(4)

免疫細胞表面標記分析

(5)

參與免疫反應的細胞

(6)

參與免疫反應的細胞

Primary organs

bone marrow

thymus

Secondary organs and tissues

spleen

lymph nodes

Peyer's patches

(7)

參與免疫反應的組織

spleen lymph node

(8)

LYMPHOCYTE SURFACE ANTIGENS

IMMUNOGLOBULINS: "Antibodies"

secreted by or found on B-Cells.

Has a huge range of specificities achieved by DNA rearrangement.

Five general types (see below): IgG, IgA, IgE, IgD, IgM

(9)

LYMPHOCYTE SURFACE ANTIGENS

T-CELL (TcR) RECEPTORS: They bind to

the Antigen-Presenting Cell.

VARIABLE REGIONS are on the T-Cell

Receptor. They allow us to develop variability and diversity in the immune response.

"CD" ANTIGENS: Systematic

classification of surface-antigens with

diverse functions. Cell-surface markers.

(10)

Cluster of Differentiation (CD) Antigens

Leukocytes express distinct assortments of molecules on their cell surfaces

many of which reflect either different stages of their lineage-specific

differentiation

different states of activation or inactivation

(11)

Cell surface immunophenotypes

different leukocyte subpopulations, including

the functionally distinct mature lymphocyte subpopulations

B-cells

helper T-cells (TH)

cytotoxic T-cells (TC)

natural killer (NK) cells

(12)

Antigen markers on mature

lymphocyte populations

(13)

Conventions for Naming Leukocyte Surface Molecules

Named according to a particular function affected by an anti-leukocyte mAb

the lymphocyte function-associated antigen 1, or LFA-1, was so named because antibodies recognizing this structure interfere with lymphocyte cell adhesion events and optimal lymphocyte function.

According to individual laboratory preferences.

B7 and B220, except that the leading "B" reminds us that these antigens are typically expressed on B lymphocytes.

Named systematically by assigning them a cluster of differentiation (CD) antigen number

identical unique reactivity pattern with different leukocyte populations.

(14)

CD antigens have also been named by one of the other conventions

CD54 = LFA-1

is widely expressed on a variety of haematopoietic cells

CD80 = B7 (or now B7-1)

CD45 = B220

CD4 = L3T4; W3/25

expressed almost exclusively on T helper (TH) lymphocytes and cells of the

monocyte/macrophage lineage

(15)

Table of CD Antigens by the NIH

http://mpr.nci.nih.gov/prow/

(16)

Cluster of Differentiation information

PROW:

Protein Reviews On the Web is an online resource that features PROW Guides

IWHLDA:

International Workshops on Human Leukocyte Differentiation Antigens

8th International Conference on Human Leucocyte Differentiation Antigens

Adelaide, South Australia 12-16 December 2004

(17)

The GUIDE of PROW and IWHLDA

(18)

CD antigen - Function

(19)

CD antigen - Structure

(20)

CD antigen –Molecular interaction

(21)

CD antigen –Expression

(22)

CD antigen –Reagent

(23)

Leukocyte subpopulation

T lymphocyte

CD1~8, CD27, CD28, CD38, CD39, CDw60, CD45, CD45RA, CD45RB, CD45RO, CD98, CD99, CD99R, CD100, CDw101

B lymphocyte

Cd10, CD19~24, CD37, CD40, CD53, CD72~75, CDw76, CD77, CD78, CD79a, CD79b, CD80~83, CDw84, CD85, CD86

Dendritic cells

CD4, CD8, CD11c, CD13, CD80, CD86, CD123, CD205, CD209, B7-DC, TLR3

(24)

Leukocyte subpopulation

Monocyte/Macrophage

CD11b, CD13, CD14, CD80, CD86, CD115, Mac-3, TLR2, TLR4

Myeloid cells

CDw12, CD13~w17, CD32~35, CD64, CDw65, CD66a~68, CD87~93

NK

CD11b, CD56, CD57, CD59, CD94, NK1.1, PanNK

Platelet

CD9, CD31, CD36, CD41a, CD41b, CD42a~42d, CD61, CD63, CD107a, CD107b

(25)

Leukocyte subpopulation

Activated antigen

CD25, CD26, CD30, CD69~71, CD95~97

Adhesion molecular

CD11a~11c, CD15s, CD18, CD29,

CD43~44R, CD48, CD49a~49f, CD50,

CD51/61, CD54~55, CD59, CD62E, CD62L, CD62P, CD102~104, CDw108

Endothelial cells

CD105, CD106, CDw109

(26)

Leukocyte subpopulation

Epithelial cells

CD104, CD133

Cytokine receptor

CD25, CD115, CDw116, CD117, CDw119, CD120a, CD120b, CDw121a, CDw121b,

CD122, CDw124, CD126, CDw127, CDw128, CDw130

Toll-like receptors

TLR1~10

(27)

Subsets of Lymphocytes

10 Rare

10 CD16 (Fc receptor for IgG);

CD3 αβ heterodimers (Limited

specificity for glycolipid-CD1 complexes)

Suppress or activate innate and adaptive immune responses NKT cells

10 Rare

10 CD16 (Fc receptor for IgG) Various activating and

inhibitory receptors

Limited specificities for MHC or MHC-like molecules Killing of virus-infected or damaged

cells (innate immunity) Natural killer

cells

40-45 20-25

10-15 Fc receptors; class II MHC;

CD19; CD21 Surface antibody

Diverse specificities for all types of molecules Antibody production (humoral

immunity) B lymphocytes

CD3+, CD4, and CD8 variable

γδ heterodimers Limited specificities for peptide and nonpeptide antigens

Helper and cytotoxic functions (innate immunity)

γδ T lymphocytes

10 10

Rare CD3+, CD4+, CD25+(Most

common, but other phenotypes as well) αβ heterodimers

Suppress function of other T cells (regulation of immune responses, maintenance of self-tolerance) Regulatory T

cells

10-15 15-20

20-25 CD3+, CD4-, CD8+

αβ heterodimers Diverse specificities for peptide-class I

MHC complexes Killing of cell infected with microbes,

killing of tumor cells CD8+cytotoxic

T lymphocytes

50-60 50-60

50-60*

CD3+, CD4+, CD8- αβ heterodimers

Diverse specificities for peptide-class II

MHC complexes B cell differentiation (humoral

immunity)

Macrophage activation (cell- mediated immunity)

CD4+helper T lymphocytes

Spleen Lymph

node Blood

αβ T lymphocytes

Percent of total lymphocytes (human)

Selected markers Antigen receptor and

specificity Functions

Class

*In most cases, the ratio of CD4+CD8-to CD8+CD4- cells is about 2:1.

Abbreviations: IgG, immunoglobulin G; MHC, major histocompatibility complex

(28)

Small Large; more cytoplasm

Small; scant cytoplasm Morphology

CD45RO; variable CD45RO

CD45RA Major CD45 isoform

(humans only)

Variable Low

High Chemokine receptor:

CCR7

High High

Low Adhesion molecules:

integrins, CD44

Low or variable Low

High Peripheral lymph node

homing receptor (L- selectin, CD62L)

Low High

Low High-affinity IL-2 receptor

Surface protein expression

+/- Yes

No Cell cycling

None Cytokine secretion;

cytotoxic activity None

Effector functions

Low High

Very low Frequency of cells

responsive to particular antigen

Preferentially to inflamed tissues, mucosal tissues Preferentially to inflamed

tissues Preferentially to peripheral

lymphoid tissues Migration

T lymphocytes

Memory lymphocytes Activated or effector

lymphocytes Naive lymphocytes

(29)

High High

Low CD27

? Low

High Chemokine receptor:

CXCR5

Small Large; more cytoplasm;

some are plasma cells Small; scant cytoplasm

Morphology

None Antibody secretion

None Effector function

Relatively high Increases during immune

response Relatively low

Affinity of Ig produced

Frequently IgG, IgA, IgE Frequently IgG, IgA, IgE

IgM and IgD Membrane immunoglobulin

(Ig) isotype B lymphocytes

Memory lymphocytes Activated or effector

lymphocytes Naive lymphocytes

(30)

Migration of naive and effector T

lymphocytes

(31)

Anti-CD marker w/w Fluorochrome

Fluorochrome

Excitation: UV, Argon-ion laser, Diode

340 nm, 488 nm, 635 nm

Emission:

FL1: 530/30 nm, FITC, GFP

FL2: 585/42 nm, PE, PI

FL3: 650 nm, 7-AAD, PerCP, PE-Cy5

FL4: 661/16 nm, APC, APC-Cy7, TOTO-3

(32)
(33)
(34)
(35)
(36)
(37)

Handling, Storage, and Preparation

of Human Blood Cells

(38)

Whole blood analysis

RBC lysis (50 lwhole blood)

Hypotonic shock-1

[9]: H2O→[1]: 10x DPBS

[5]: 0.1x HBSS→[5]: 2x HBSS

10x ammonium chloride lysis solution

89.9 g NH4Cl

10.0 g KHCO3

370.0 mg tetra-sodium EDTA

Adjust to pH 7.3. Store at 2 to 8 deg. C in a tightly closed bottle

1000~1500 events/second in Hi speed (60 l/min)

(39)

Whole blood analysis

lymphocytes

monocytes granulocyte

(40)

Purified mononuclear cells

Ficoll-PaqueTM PLUS

Dilute whole blood with HBSS (PBS, DPBS)

Ficoll-Paque [3] + dilute blood [4]

600~800 xg in RT (25~18oC) for 30 min w/o brake

VACUTAINER CPT (Cell Preparation Tubes,

BD Cat. No. 362753, 362761)

HISTOPAQUE (Sigma)

1077 for human

1083 for rat, mouse

1119 for separate MNC and neutrophils

(41)

VACUTAINER CPT, 1500~1800 xg

(42)

Purified mononuclear cells

lymphocytes

(43)

Purified mononuclear cells

monocytes for CD14

(44)

Purified mononuclear cells

lymphocytes/monocytes

(45)

Spleen analysis

(46)

Spleen analysis

Hypotonic shock

Shaking time

For NK activity analysis

Cell surface marker staining

1x107/ml → take 50 l(5x105 cells)

w/o fix cells

Resuspend in HBSS containing NaN3 and 2% FBS

800~1000 events/sec in Hi speed (60 l/min)

(47)

Spleen analysis-CD4/CD25

8.39%

3.44%

4.27%

(48)

Spleen analysis-CD8/CD25

(49)

Lymph node analysis

(50)

Lymph node analysis- CD4/CD25

5.31%

4.19%

(51)

Lymph node analysis-CD3/4/8

34.06%

27.42%

29.13%

31.96%

(52)

Lymph node analysis- CD3/19/45

22.46%

75.52%

76.95%

23.89%

(53)

Thymus analysis-CD4/25/69

(54)

Thymus analysis-CD3/4/8

(55)

Thymus analysis-CD3/19/45

(56)

Intracellular cytokines

analysis

(57)

The Th1/Th2 paradigm

Sergio Romagnani.

Immunology Today.

18: 263-266. 1997.

(58)

Th2 cell in allergic disease

Lewis, David B.

Curr. Opin. Immunol.

14: 644-651. 2002

N Novak. et al., Allergy. 54:

792-803. 1999

(59)

Jane L Grogan and Richard M Locksley. Curr. Opin. Immunol. 14: 366-372. 2002.

Helper T cell differentiation

(60)

Jane L Grogan and Richard M Locksley. Curr. Opin. Immunol. 14: 366-372. 2002.

Helper T cell polarization

(61)

Alessandra B. Pernis and Paul B. Rothman. J. Clin. Invest. 109:1279–1283 (2002).

JAK-STAT signaling in asthma

(62)

Th2 activate B cell

Alessandra B. Pernis and Paul B. Rothman. J. Clin. Invest. 109:1279–1283 (2002).

(63)

Analysis approach

Environments

Ag stimulation → ELISA

Signaling gene expression

Northern

Western

PCR, Real Time-PCR

Intracellular cytokine detection

FACS

(64)

Allergic disorder

Asthma

Dermatitis

Autoimmune disease

(65)

Stimulation of Cells

Activation

50 ng/ml of PMA (Phorbol-12-myristate-13-acetate), + 1 M of ionomycin, or 250 ng/ml calcium ionophore A23187

ConA (3-5 g/ml)

6-48 hr

Re-stimulation

anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr

Inhibit intracellular cytokine transport

1-3 M monensin, or 1-5 g/ml brefeldin A

4-6 hr

(66)

Annotations: mouse PEC=mouse thioglycolate-elicited peritoneal macrophages; ConA=Concanavalin A; Iono=Ionomycin; LPS=Lipopolysaccharide; PMA=Phorbol Myristate Acetate; 2d=2 day culture; 5hr=5 hour culture

TN3-19.12 Monensin

anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr

2d/3d ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4

(20ng/ml) (3d) mouse

spleen TNF-a

MP6-XT22 Monensin

anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr

2d/3d ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4

(20ng/ml) (3d) mouse

spleen TNF-a

XMG1.2 Monensin

anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr

2d/3d ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4

(20ng/ml) (3d) mouse

spleen IFN-g

MP1-22E9 Monensin

anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr

2d/3d ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4

(20ng/ml) (3d) mouse

spleen GM-CSF

C17.8 Monensin

- 2hr/22hr mIFNg (100ng/ml) (2hr)/LPS (100ng/ml)

(22hr) mouse

IL-12 PEC

JES5-16E3 Monensin

anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr

2d/3d ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4

(20ng/ml) (3d) mouse

spleen IL-10

MP5-20F3 Monensin

anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr

2d/3d ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4

(20ng/ml) (3d) mouse

spleen IL-6

BVD6-24G2 Monensin

anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr

2d/3d ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4

(20ng/ml) (3d) mouse

spleen IL-4

JES6-5H4 Monensin

anti-CD3 (10ug/ml immobilized)+anti-CD28 (2ug/ml soluble) 5hr

2d/3d ConA (3ug/ml) (2d)/IL-2 (20ng/ml)+IL-4

(20ng/ml) (3d) mouse

spleen IL-2

B122 Monensin

- 2hr/22hr mINFg (100ng/ml)(2h4)/LPS

(100ng/ml)(22hr) mouse

IL-1b PEC

ALF-161 Monensin

- 2hr/22hr mINFg (100ng/ml)(2h4)/LPS

(100ng/ml)(22hr) mouse

IL-1a PEC

Antibody Intracellular

Block Restimulation

Incubation Activation Time

Cell Source Mouse

Cytokine

Mouse Cytokine Intracellular Staining Quick Guide

(67)

Annotations: Iono=Ionomycin; PMA=Phorbol Myristate Acetate; LPS=Lipopolysaccharide; 2d=2 day culture; 5hr=5 hour culture; LPS for activation of human PBMC obtained from Sigma (#L-8274)

MAb11 Monensin

- PMA (30-50ng/ml)/Iono 5hr

(1ug/ml) PBMC

TNF-a

4S.B3 Monensin

- PMA (30-50ng/ml)/Iono 5hr

(1ug/ml) PBMC

IFN-g

C8.6 Monensin

- 2hr/22hr

hIFNg (100ng/ml) (2hr)/LPS (100ng/ml) (22hr)

PBMC IL-12

JES3-9D7 Monensin

- 24hr

LPS 100ng/ml PBMC

IL-10

MQ2-13A5 Monensin

- 5hr

LPS 100ng/ml PBMC

IL-6

MP4-25D2 Monensin

PMA (5ng/ml) + Ionomycin (500ng/ml) (4hr) 2d/3d

anti-CD3 (10µg/ml, immobilized) + anti- CD28 (2µg/ml, soluble) + IL-2 (10ng/ml) + IL-4 (20ng/ml) (2d); IL-2 (10ng/ml) + IL-4 (20ng/ml) (3d) PBMC

IL-4

MQ1-17H12 Monensin

- PMA (30-50ng/ml)/Iono 5hr

(1ug/ml) PBMC

IL-2

CRM56 Monensin

- 24hr

LPS 100ng/ml PBMC

IL-1b

CRM8 Monensin

- 24hr

LPS 100ng/ml PBMC

IL-1a

Antibody Intracellular

Block Restimulation

Incubation Activation Time

Cell Source Human

Cytokine

Human Cytokine Intracellular Staining Quick Guide

(68)

Flurochrome-conjugated staining surface antigen for T helper cells

106 cells in 100 lof staining buffer

mAb CD3-PerCP → T cell

CD4-FITC → T helper cell

30 min, 4 °C in dark

Staining buffer

DPBS without Mg2+ or Ca2+

1 % FBS

0.1 % NaN3

Adjust pH 7.4-7.6, filter, store at 4 °C

(69)

Fix the cells

4 % (w/v) paraformaldehyde in DPBS with 0.54 % glucose

20 min. at 4°C

Cell can be kept overnight in fixation buffer at 4°C in dark

(70)

Permeabilize cells

Wash cells 2 times in permeabilization buffer and pellet

permeabilization buffer:

DPBS

1 % FBS

0.1 % (w/v) NaN3

0.1 % (w/v) saponin

0.1 % glucose

0.01 M HEPES

0.035 % NaHCO3

Adjust buffer pH to 7.4-7.6 and filter

(71)

Stain intracellular cytokines

Resuspend fixed cells in 100 lof permeabilization buffer

mAb anti-IFN-,anti-IL-4, anti-TNF-

Incubate at 4°C for 30 min. in dark

(72)

Analysis

Resuspense cells in staining buffer

Set PMT voltage and compensation

IFN-

(73)

Analysis

Resuspension cells in staining buffer

Set PMT voltage and compensation

IL-4

(74)

Analysis

Resuspension cells in staining buffer

Set PMT voltage and compensation

TNF-

(75)

Analysis

Resuspension cells in staining buffer

Set PMT voltage and compensation

IL-4 and IFN-double stain

(76)

流式細胞多重分析技術

Cytometric Bead Array, CBA

細胞激素分析的方法乃是利用抗體標定微球吸附

細胞培養上清液中之細胞激素,在利用流式細胞 儀分析細胞激素所呈現出來之不同螢光讀值,再 與標準曲線對照而得其分泌量

(77)
(78)
(79)

Platelet analysis

(80)

Platelet analysis-Scatter Gating

(81)

Platelet analysis-Fluorescence Gating

(82)

Platelet analysis

ADP, 2 min.

Control

(83)

Surface markers of platelet of platelet activation.

PAC-1 CD62P

CD61 CD61

Modified from: Kestin et al. Circulation (1993) 88(4 Pt 1): 1502-1511.

(84)

活化血小板測定意義

抗體之選用:

PAC-1 ( 結合活化血小板膜上之GPIIb/IIIa)

CD62P ( 結合活化血小板膜上之P-selectin)

CD61 ( 結合靜止及活化血小板膜上之GPIb/IX)

PAC-1 / CD61 :

活化血小板數目/ 所有血小板數目

觀察抗氧化補充劑對血小板膜蛋白之影響

CD62P / CD61:

活化血小板數目/ 所有血小板數目

觀察抗氧化補充劑對血小板- granule膜蛋白之影響

(85)

A. B.

D.

C.

Platelet

CD62P+/ PAC-1+ 2.85%

PAC-1+ 4.84%

CD62P+ 19.24%

CD62P+ 17.21%

Platelet

Flow Cytometry Human Platelet Activation Analysis

(86)
(87)
(88)
(89)
(90)
(91)
(92)

Insulin resistance

Cell: FL83B (mouse hepatocyte, BCRC 60325)

2-NBDG (MW. 342.26, Invitrogen)

Ex: 487 nm, Em: 542 nm

(93)

(Etxeberria et al., 2005)

(94)

Kojima et al., 2010)

(95)

(Presley et al., 2010)

(96)

Regulation of Hematopoiesis by Cytokines - Human

http://cgap.nci.nih.gov/Pathways/BioCarta/stemPathway

(97)

Regulation of Hematopoiesis by Cytokines - Mouse

http://cgap.nci.nih.gov/Pathways/BioCarta/m_stemPathway

(98)

Apoptosis analysis

(99)

細胞凋亡

細胞凋亡又稱為程序化細胞死亡,是某些

生理或病理條件下,細胞接受某種信號的 觸發後主動發生一連串連續性細胞變化,

最終導致細胞死亡而不引起炎症的細胞死 亡過程。細胞凋亡現象是由 Kerr Whyuie 等人於1972 年首次提出,1980 年他們在 對細胞凋亡進行長期觀察和分析後提出細 胞凋亡不同於細胞壞死。

(100)

細胞凋亡的生物學意義

細 胞 凋 亡 被 認 為 是 與 細 胞 增 生 相 反 的 方 式 , 來 調 節 細 胞 群 體 , 它 不 僅 對 胚 胎 發 生、器官發育、分化作用、及保持機體的 平衡穩定等過程至為重要,而且對控制細 胞的增殖、腫瘤發生和發展極為重要。通 過細胞凋亡,機體及時清除受損及危險的 細胞,因此細胞凋亡對機體的正常發展具 有十分重要的生物學意義。

(101)

細胞凋亡的生物學意義

細胞凋亡可經由

Deprivation of growth factors

-irradiation

Oxygen free radical (OFR) production

Receptor-ligand interaction

Inhibition protein kinase

(102)

凋亡細胞的特徵

細胞會喪失微絨毛、偽足等胞膜結構,隨後,細

胞會皺縮、核質濃縮,細胞密度增大。

細胞核離散,呈現月牙形凝集在核膜下。

隨著細胞膜和細胞核分離,會裂解形成凋亡小體

(Apoptotic body)。

凋亡過程的最後階段發生細胞核的 DNA 降解,

降解後產生的 DNA 片段由185-200 bp 多聚體組 成。在瓊脂凝膠上呈現特徵性凋亡“梯型”。

(103)

凋亡細胞的特徵

(104)

程序性死亡

程序性死亡 - - 凋亡的活化 凋亡

粒線體

膜電位的失衡

Apaf-1/cyt. c/AIF release

Bcl family regulation

TNFR/FAS superfamily

P53/Rb cell cycle checkpoint pathway

Toll-Like receptor pathway

Nicotinic acetylcholine receptors pathway

Cell-cell interaction

Growth factor/cytokine pathway

(105)

程序性死亡

程序性死亡 - - 凋亡程序訊息傳遞 凋亡

Caspase pathway (Mit., TNFR, P53…)

PI3K-Akt pathway

Activate cytokine/growth factor release

(106)

程序性死亡-DNA fragmentation

Endo G

CAD/DFF

Lamin B1/Lamin degradation

(107)

粒腺體在凋亡程序中所扮演的角色

能量代謝失衡

Produce OFR

Alter the redox state of the cell

Cause cycling of Ca2+ ions

Release cyt c, Apfa-1, AIF

Regulate protooncogene Bcl-2 family

Release endonuclease

(108)

粒腺體在凋亡程序中所扮演的角色

(109)

粒腺體膜電位的偵測

5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; CBIC2(3))

(110)

粒腺體膜電位的偵測

NIH 3T3 fibroblasts stained with JC-1 showing the progressive loss of red J-aggregate fluorescence and

cytoplasmic diffusion of green monomer fluorescence following exposure to hydrogen peroxide.

Images show the same field of cells viewed before H2O2 treatment, and 5, 10 and 20 minutes after treatment.

(Images contributed by Ildo Nicoletti, Perugia University Medical School.)

(111)

粒腺體膜電位的偵測

Bivariate JC-1 analysis of mitochondrial membrane potential in HL60 cells by flow cytometry. The sensitivity of this technique is demonstrated by the response to valinomycin-induced depolarization for two hours. Figure courtesy of Dr Andrea Cossarizza, University of Modena and Reggio Emilia.

(112)

粒腺體膜電位的偵測

(113)

粒腺體能量代謝異常-PS presentation

Figure from North American Scientific, Inc.

http://www.nasi.net/ProductCenter/mi/mi_howapoptosis.html

(114)

粒腺體能量代謝異常

-PS presentation/Annexin V-FITC/PI

Biotechnol. Appl. Biochem. 2001, 33: 127–132

(115)

粒腺體能量代謝異常

-PS presentation/Merocyanine 540/7-AAD

(116)

細胞凋亡-活化TNFR superfamily路徑

(117)

細胞凋亡-活化TNFR superfamily路徑

(118)

Caspase pathway

(119)

Caspase pathway-reagents

Ac-LEHD-AFC FAM-LEHD-FMK

Ac-LEHD-AMC Ac-LEHD-pNA

Caspase 9

Gentaur

FAM-LETD-FMK FAM-YVAD-FMK FAM-VEID-FMK FAM-DEVD-FMK (E/M=490/520)

Fluorimetric-2

Roche Sigma

Sigma

Ac-LETD-AFC Ac-LETD-AMC

Ac-LETD-pNA Caspase 8

Ac-YVAD-AFC Ac-YVAD-AMC

Ac-YVAD-pNA Caspase 1

Ac-VEID-AFC Ac-VEID-AMC

Ac-VEID-pNA Caspase 6

Ac-DEVD-AFC (E/M=400/505) Ac-DEVD-AMC

(E/M=360/460) Ac-DEVD-pNA

(405) Caspase 3

Fluorimetric-3 Fluorimetric-1

Colorimetric Caspase

(120)

APO 2.7 detection

(121)

Tubulin/PI stain

(122)

TUNEL Assay

(123)

PI Stain for analysis pre-G0 peak

Control 10.3%

38.9% 64.7%

(124)

DNA fragmentation

(125)

上樣與資料分析

(126)

A single laser flow cytometer

(127)

A dual laser flow cytometer

(128)

A dual laser flow cytometer

(129)

Fluorochromes

(130)

Fluorochromes

(131)

FACS 日常操作

儀器本體,及Macintosh電腦。

a. 電源:電源在儀器右側下方,操作時要先啟 動儀器本體再打開電腦及印表機。

b. 暖機時間:儀器需5 ~10分鐘的暖機時間

c. 儀器面板:

流速控制鍵(LO/MED /HI)

功能控制鍵(BACKFLUSH/RUN/STANDBY/ PRIME- DRAIN/FILL)

(132)

FACS 日常操作

流速控制:

LO: 樣品流速:12 l/min

MED: 樣品流速:35 l/min

HI: 樣品流速:60 l/min

功能控制:

RUN: 綠色時表示樣品開始輸注。(黃色時表示儀 器不正常,請檢查是否漏氣)

STANDBY: 無樣品或暖機時之正常位置,此時雷 射功率會自動降低以延長雷射壽命。

PRIME: 自動沖洗進樣針並將PBS 注滿Flow

Cell,使用時機如:裝機開機、更換PBS、清洗儀 器或清洗進樣針等。

(133)

FACS 開機

開啟細胞儀電源。

開啟其他周邊配備電源,如印表機及M.O.。

開啟電腦。

確認鞘流液筒有八分滿的FACS FLOW,確實旋緊。

將廢液倒掉,並在廢液筒中加入100 ml 家用漂白水。

將氣壓閥方向調在加壓(Pressurize)位置。

排除液流過濾氣中的氣泡。

使用1 ml PBS 為樣品,執行PRIME 功能兩次。

HIGH RUN 兩分鐘,即可開始分析樣品。

(134)

檢品上機之確認事項

是否已將檢品濃度調至1X106 cells/ml?

是否已去除檢品中之細胞團塊,以防止管路堵塞?可使用附濾網 FALCON試管(Cat. No.2235) 或30-50 m 的尼龍篩網。

是否已將檢品放至FALCON 2052 試管中?試管是否有裂痕?

是否已將專用鞘流液筒充填至八分滿?

是否已將廢液倒掉,並在廢液筒中加入100 ml 漂白水?

是否已將液流過濾器中之氣泡排空?

是否已將所有管線及管路裝置妥善?並將氣壓閥方向調至正確定 位?

是否已執行Prime 兩次以將管路及Flow Cell 中之氣泡排空?

填寫使用登記表。

(135)
(136)

Compensation

(137)
(138)
(139)
(140)
(141)
(142)
(143)

FACS 關機

執行「日常除污」與「日常清洗」時機:

如上機樣品含特殊染劑(如DNA/RNA核酸染 料),需執行「日常除污」與「日常清洗」。

全血樣品如進行Lyse w/o Wash分析(如CD34 Stem Cell),需執行「日常除污」與「日常清 洗」

一般細胞株、或全血樣品進行Lyse-Wash分 析,只需執行「日常清洗」。

(144)

“日常除污”程序 (FACS Rinse)

將樣品支持架左移。

取2 ml FACS Rinse 上樣品,讓儀器的真空系 統抽取約1 ml 的液體。

將樣品支持架回正,執行PRIME 功能兩次,

按HI RUN,然後讓FACS Rinse 清洗管路5分 鐘。

取2 ml Milli-Q 上樣品,重覆上述步驟1-3。

FACS Rinse: 0.5 % Triton X-100 in Milli-Q

(145)

“日常清洗”程序 (FACS Clean)

將樣品支持架左移。

取2 ml FACS Clean 上樣品,讓儀器的真空系 統抽取約1 ml 的液體。

將樣品支持架回正,執行PRIME 功能兩次,

按HI RUN,然後讓FACS Rinse 清洗管路5分 鐘。

取2 ml Milli-Q ,重覆上述步驟1-3。

注意最後只留約1 ml Milli-Q 在試管中。

FACS Clean: 10 % (5 %)漂白水

(146)

FACS Calibur 關機

按Standby 以冷卻雷射,Standby五分鐘後關 閉細胞儀。(務必等五-十分鐘後再關

FACSCalibur電源,以延長雷射光源壽命。)

倒掉廢液,並回填100 c.c.漂白水。

將氣壓閥放在「漏氣」位置。

確認退出電腦中BD應用軟體,數據資料已儲

存備份。關程式“File”-“Quit”(選擇“Don‘t save”)

關閉蘋果電腦。“Special”-“Shutdown”。

(147)

FACSCalibur

(148)

FACSAria

Argon-Ion Laser 488nm、He-Ne Laser 633nm,可分析7色螢光

多色螢光分析 分選

細胞功能性分析

稀有細胞分析 分選

造血幹細胞分析 分選

依據核酸含量分選腫瘤細胞

(149)

FACSAria

(150)

相關資訊

流式實驗指南

流式分析技術 (臨床診斷應用、基礎醫學研究、生 技製藥應用)

螢光染劑與多色流式細胞分析

流式細胞應用--細胞存活之檢測

BD FACSAria Sorting注意事項

BD FACSAria清洗與滅菌事宜

Current protocol in cytometry, CP search

Protocol-online

Google-Scholar, Scirus

(151)

謝謝

謝長奇

http://web.thu.edu.tw/cchsieh/www/

cchsieh@thu.edu.tw

TEL: 04-23590121#37125, 37100

Figure

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References

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