Microsoft PowerPoint - 프레젠테이션2

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1 Apoptosis Products

2 Apoptosis & Necrosis * Apoptosis (programmed cell death) 세포가유전자에의해제어되어죽는방식의한형태 * Necrosis 화상과타박, 독물등의자극에의해생체세포, 조직이일부가죽거나죽어가는상태

3 Apoptosis & Necrosis Apoptosis Active cell death DNA cleaved, chromatin condenses Cells shrink Formation of apoptotic body Cleared by phagocytosis No inflammation = no tissue damage Necrosis Passive cell death Cells swell up Membrane breaks down and cellular contents Nucleus disintegrates Cell ghosts Inflammatory=tissue damage

4 Two Different Types of cell death

5 Apoptosis 의특징및관찰방법 DNA 의 fragment - 핵의 double strand DNA 가절단되어 180~200base pair 길이의배수로절단된조각이만들어짐. - Agarose gel EP 에서특징적인 ladder pattern 의 band 형태 - Necrosis 에서나타나는무작위적인 DNA 분해에의한 diffuse smear pattern 과비교 - Agrose gel EP - TUNEL Method : 말단표식법에의해조직화학적으로검출하는 kit apoptosis 를일으킨세포에서단편화된 DNA 의유리 3'-OH 말단을 terminal transferase(tdt) 로 fluorescein-dutp 를고효율적이면서도특이적으로표식한후형광현미경이나 flow cytometry 로검출하는방법 Apoptotic body - 단백질의 cross-linking을일으켜세포막주위에딱딱한껍질을형성하여 apoptotic body가탐식되기전에내용물이빠져나오지못하게하는역할 - phosphatidylserine(ps) 가노출된형태 - phagocyte 에는 vitronectine 수용체와같은수용체가있어 apoptotic p body 를인지하고탐식한다 - Annexin V 이용 - Annexin V binds to PS at 1mM ca2+(calcium dependent manner) - Annexin V에여러형광들을붙여 flow cytometer나형광현미경을통해서관찰.

6 Apoptosis 의관찰방법 * Apoptosis 의 interpretation Annexin V TUNEL interpretation positive negative early execution phase of apoptosis positive positive late execution phase of apoptosis negative positive cells located in phagolysosomes

7 Biovision Product Annexin V DNA fragmentation assay kit Caspase Assay kit Mitochondrial Apoptosis Kit Calpain Assay Kit Cathepsin assay kit sirna vector

8 Annexin V Annexin V conjugates Flow Chart of the Annexin V Assay Collect cells by centrifugation Add annexin V conjugate in 1x binding buffer Incubate 5min in the dark Analyze by flow cytometry or fluoresence microscopy Various Annexin V Conjugates are available for detecting phosphatidyl h serine (PS) exposure in apoptotic cells.

9 Annexin V Annexin V-FITC, K101 & K201 Green color : flow cytometry (ex/em=488nm/530nm) or fluorescence microscopy(dual filter set for FITC and rhodamine K101 Propidium Iodide(PI) K201 SYTOX Green Dye : FL1 channel Annexin V-EGFP, K104 * PI ( 붉은색 ): necrosis cell 에염색 Green color : flow cytometry (ex/em=488nm/530nm) or fluorescence microscopy(dual filter set for FITC and rhodamine Brighter and more photo stable than other fluorescent reagents Annexin V-Cy3, K102, K202 Red color : flow cytometry (ex=488nm or 543nm;em 570nm) 570nm) or fluorescence microscopy(rhodamine filter) For Double staining with GFPexpressing cells K202 : SYTOX green Dye Annexin V-Cy5, K103 Blue color : flow cytometer (ex=649nm;em=670nm) or fluorescence microscopy(cy5 filter) For triple staining of a cell, in conjunction with green and red fluorescence staining

10 Annexin V Annexin V-PE, K128 Orange red color : Flow cytometry(ex=488;em=578), y fluorescence microscopy(rodamine filter) Annexin V-PE-Cy5, K129 Purple color : Flow cytometry (ex=488;em=670), fluorescence microscopy(pe Cy5 filter)) Annexin V-Biotin, K109 dye staining using any streptavidine or avidin dye reagents, such as (strept)avidinefluorescein, peroxidase, alkaline phosphatase (AP), and β gal, etc.

11 DNA Fragmentation Assay Kits Apoptotic p DNA Ladder Detection Kits 핵의 double strand DNA 가절단되어 180~200base pair 길이의배수로절단된조각이만들어짐 TUNEL based DNA Fragmentation Assay Kits apoptosis 를일으킨세포에서단편화된 DNA 의유리 3' OH 말단을 terminal p p 3 transferase(tdt) 로 fluorescein dutp 를고효율적이면서도특이적으로표식한후형광현미경이나 flow cytometry 로검출하는방법

12 Apoptotic ti DNA Ldd Ladder Dt Detection ti Kits Quick Detection of Apoptotic DNA Ladder in Jurkat Cells. Apoptosis was induced in Jurkat cells with camptothecin (2 mm) for 0 hr (Lane 1), 6 hrs (Lane 2) and 12 hrs (Lane 3). Chromosomal DNA was prepared using the Quick Apoptotic DNA Ladder Detection Kit according to the kit instructions. 20 ml of each sample was electrophoresed on a 1.2% agarose/etbr gel. K120 : Quick Apoptotic DNA Ladder Detection Kit 90 minutes to prepare chromosomal DNA K130 : Enhanced Apoptotic DNA Ladder Detection Kit staining Dye -To improves detection sensitive K170 : Apoptotic DNA Ladder Isolation Kit detect from Tissue and Cell, 5% apoptotic cell

13 TUNEL based DNA Fragment Assay Kits

14 DNA Fragmentation Assay Kits * Cell 에 GFP 가첨가되어있는경우

15 Apoptosis p Pathway 형태학적, 생화학적인변화를기준으로보면크게 3 stage ( 초기, 중기, 말기 ) 로나누어짐. - 초기 : 탈수에의해세포크기가줄어듬. DNA 분해가시작, 세포내칼슘의농도증가. -> 세포막안쪽으로노출되는 Phosphatidylserine (PS) 막지질이세포막바깥쪽으로노출 - 중기 : DNA가 180~200bp정도의단편화이루어짐 (DNA 전기영동상 Ladder 형태로보임 ) -> 조각난 DNA와세포질일부를포함하는 apoptotic body 형성 - 말기 : 세포막의기능이완전소실되어식세포에의해포식. Apoptosis Inducing Factor (AIF) - Extrinsic i pathway : Death 신호와 death 수용체와결합해세포내부로신호를전함. -> procaspase가 caspase로활성화시켜 apoptosis가일어남. - Intrinsic pathway : 발달과정에서유래하는세포신호혹은세포 domage에의해반응하여세포질내 Bcl-2 family protein 의활성화 -> 막전위상실, mitochondrial outermembrane permeabilization 를유발하여안에있던 cytc 가세포질로흘러들어가 Apaf1/cytc/ATP으로구성되어 caspase 9을활성화하여 apoptosis가일어남.

16 Apoptosis Pathway

17 Caspase Study Tools Caspase -cystein protease 로써세포를구성하는단백질을분해 y p 써세구성하백해 -aspartate (D) xxx bond 를분해 - Caspase 1~12 종까지현재알려져있다.

18 Caspase Assay Kit CasGLOWTM In Situ Caspase 2,3,8,9,12 Staining Kits Caspase inhibitor

19 Caspase Assay Kit Caspase Inhibitor Drug Screening Kits Kit components : casapase substrate Active Human Caspase Caspase Inhibitor Caspase 를저해할수있는물질개발연구에적용가능

20 Caspase Assay Kit Fluorometric &C Colorimetric i caspase Assay - Fluorometric : Substrate AFC -> Fluorometer 사용 ( 형광 ) - Colometric :Substrate pna -> spectrophotometer 사용 ( 발색 ) CaspSCREEN TM Apoptosis Detection Kit - Living cell에서 flow cytometry에의해 caspase 활성을보기위해사용 - caspase family 의기질로써 (aspartyl)2-rhodamin 110(D 2 R) 의분해에의해측정 CaspSELECT TM Caspase-3 and -7 Assay Kit - 똑같은 substrate sequence (DEVD) 에 target 을하기때문에각각의활성을 detection 하기어렵다. 그래서 antibody 를이용하여 ELISA 방법으로측정하는방식 그외각 inhibitor 와 substrate 를따로구매가능

21 Caspase Assay Kit RNA 단편 (21bp~23bp) 를세포내로주입하면목적 mrna 분해. Once processed, the vector expressed sirna is incorporated into a multiprotein nuclease complex known as the RNA-induced silencing complex (RISC). Homology between the sense portion of the sirna sequence and the mrna enables the nuclease enzyme to bind and cleave the caspase transcript into small pieces, which are degraded by the cell's machinery. Schematic Diagram of the RNA Interference Mechanism

22 Mitochondrial Assay Kit

23 Mitochondria mediated Proteins product Mitochondria apoptosis detection kit (K or100) - Disruption of the mitochondrial transmembrane potential - Mitocapture :a cationic dye that fluoresces(red) differently in healthy vs apoptotic cells Healthy cell -> RED: Mitocapture가 mitochondria내에응집 Apoptotic cell -> Green : Mitocapture가응집이안되고 FITC만세포질에남아있게됨 - detected by fluorescence microscopy or analyzed by flow cytometry.

24 Mitochondria mediated Proteins product Apoptosis 는 ATP 를필요로하는 energy-dependent 과정 -> ATP 양의감소는더이상대사과정을수행할수없으므로 Apoptosis 를나타낸다. ApoSENSOR TM ATP Cell Viability Assay Kit Lumimometer, beta counter 이용 ApoSENSOR ADP/ATP Ratio Assay Kit (K ) -for detection apoptosis/necrosis/growth arrest/cell proliferation simultaneously in mammalian cells - ADP convert enzyme 이용

25 Mitochondria mediated Proteins product Ct Cytochrome CA Apoptosis Assay Kit(K ) - Mitochodira 내에존재하다가 apoptosis 신호에의해외부로나옴. 정상 cell : mitochondria 내에존재 Apoptosis cell : 세포질에존재 - Mitochondria/cytosol fraction - >Cytochrom C antibody 이용 detect

26 그외 Apoptosis 에관련된 assay kit * Caipain Activity Assay Kit & Reagent * Cathepsin Assay Kits & Reagent Calpain : 단백질분해효소 Cathesin : caspase 관련분해효소

Benchtop Flow Cytometry guava easycyte Systems 머크밀리포아의 guava easycyte System 은특허받은 microcapillary 기술을이용한컴팩트한시스템으로다양하고복잡한세포분석이가능합니다. Cell Health Cell S

Benchtop Flow Cytometry guava easycyte Systems 머크밀리포아의 guava easycyte System 은특허받은 microcapillary 기술을이용한컴팩트한시스템으로다양하고복잡한세포분석이가능합니다. Cell Health Cell Signaling Stem Cells Immunology Cell Counting & Viability