流式細胞儀在
免疫細胞生物學上之應用
謝長奇
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
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
免疫細胞表面標記分析
參與免疫反應的細胞
參與免疫反應的細胞
Primary organs
bone marrow
thymus
Secondary organs and tissues
spleen
lymph nodes
Peyer's patches
參與免疫反應的組織
spleen lymph node
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
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.
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
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
Antigen markers on mature
lymphocyte populations
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.
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
Table of CD Antigens by the NIH
http://mpr.nci.nih.gov/prow/
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
The GUIDE of PROW and IWHLDA
CD antigen - Function
CD antigen - Structure
CD antigen –Molecular interaction
CD antigen –Expression
CD antigen –Reagent
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
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
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
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
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
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
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
Migration of naive and effector T
lymphocytes
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
Handling, Storage, and Preparation
of Human Blood Cells
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)
Whole blood analysis
lymphocytes
monocytes granulocyte
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
VACUTAINER CPT, 1500~1800 xg
Purified mononuclear cells
lymphocytes
Purified mononuclear cells
monocytes for CD14
Purified mononuclear cells
lymphocytes/monocytes
Spleen analysis
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)
Spleen analysis-CD4/CD25
8.39%
3.44%
4.27%
Spleen analysis-CD8/CD25
Lymph node analysis
Lymph node analysis- CD4/CD25
5.31%
4.19%
Lymph node analysis-CD3/4/8
34.06%
27.42%
29.13%
31.96%
Lymph node analysis- CD3/19/45
22.46%
75.52%
76.95%
23.89%
Thymus analysis-CD4/25/69
Thymus analysis-CD3/4/8
Thymus analysis-CD3/19/45
Intracellular cytokines
analysis
The Th1/Th2 paradigm
Sergio Romagnani.
Immunology Today.
18: 263-266. 1997.
Th2 cell in allergic disease
Lewis, David B.
Curr. Opin. Immunol.
14: 644-651. 2002
N Novak. et al., Allergy. 54:
792-803. 1999
Jane L Grogan and Richard M Locksley. Curr. Opin. Immunol. 14: 366-372. 2002.
Helper T cell differentiation
Jane L Grogan and Richard M Locksley. Curr. Opin. Immunol. 14: 366-372. 2002.
Helper T cell polarization
Alessandra B. Pernis and Paul B. Rothman. J. Clin. Invest. 109:1279–1283 (2002).
JAK-STAT signaling in asthma
Th2 activate B cell
Alessandra B. Pernis and Paul B. Rothman. J. Clin. Invest. 109:1279–1283 (2002).
Analysis approach
Environments
Ag stimulation → ELISA
Signaling gene expression
Northern
Western
PCR, Real Time-PCR
Intracellular cytokine detection
FACS
Allergic disorder
Asthma
Dermatitis
Autoimmune disease
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
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
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
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
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
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
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
Analysis
Resuspense cells in staining buffer
Set PMT voltage and compensation
IFN-
Analysis
Resuspension cells in staining buffer
Set PMT voltage and compensation
IL-4
Analysis
Resuspension cells in staining buffer
Set PMT voltage and compensation
TNF-
Analysis
Resuspension cells in staining buffer
Set PMT voltage and compensation
IL-4 and IFN-double stain
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
Platelet analysis
Platelet analysis-Scatter Gating
Platelet analysis-Fluorescence Gating
Platelet analysis
ADP, 2 min.
Control
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.
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
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
Insulin resistance
Cell: FL83B (mouse hepatocyte, BCRC 60325)
2-NBDG (MW. 342.26, Invitrogen)
Ex: 487 nm, Em: 542 nm
(Etxeberria et al., 2005)
Kojima et al., 2010)
(Presley et al., 2010)
Regulation of Hematopoiesis by Cytokines - Human
http://cgap.nci.nih.gov/Pathways/BioCarta/stemPathway
Regulation of Hematopoiesis by Cytokines - Mouse
http://cgap.nci.nih.gov/Pathways/BioCarta/m_stemPathway
Apoptosis analysis
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。
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。
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
…
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”。
凋亡細胞的特徵
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
…
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
程序性死亡-DNA fragmentation
Endo G
CAD/DFF
Lamin B1/Lamin degradation
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
粒腺體在凋亡程序中所扮演的角色
粒腺體膜電位的偵測
5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; CBIC2(3))
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.)
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.
粒腺體膜電位的偵測
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
粒腺體能量代謝異常
-PS presentation/Annexin V-FITC/PI
Biotechnol. Appl. Biochem. 2001, 33: 127–132
粒腺體能量代謝異常
-PS presentation/Merocyanine 540/7-AAD
細胞凋亡-活化TNFR superfamily路徑
細胞凋亡-活化TNFR superfamily路徑
Caspase pathway
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
APO 2.7 detection
Tubulin/PI stain
TUNEL Assay
PI Stain for analysis pre-G0 peak
Control 10.3%
38.9% 64.7%
DNA fragmentation
上樣與資料分析
A single laser flow cytometer
A dual laser flow cytometer
A dual laser flow cytometer
Fluorochromes
Fluorochromes
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) 。
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。
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。
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。
Compensation
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」。
“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
“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
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”。
FACSCalibur
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
FACSAria
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
謝謝
謝長奇
http://web.thu.edu.tw/cchsieh/www/
cchsieh@thu.edu.tw
TEL: 04-23590121#37125, 37100