Beckman Coulter???????

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• ???
• Beckman Coulter???????
• 13636440348
• dsyou_at_beckman.com

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• CYTOME ???,???????????????????
• CYTOMICS ????,???????????,???????????????????????
  ??????????????
• PROTEOME ???,?????????(???????????????????)??????
  
• PROTEOMICS ????,???????????????,???????????????

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?????
??RNA(RNAi)
??????(Cell Signalling)
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??-???-??/??? ??????
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Growth in cell based assays------??????
Past 5 years
In Development
Source Dr. Priya Kunapuli, Senior Research
Scientist, Merck Research Laboratories Dec. 2002

• Key takeaways
• Rapid movement to cell-based assays for
  contextual analysis

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18??,????????
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• ????????????

High-end automated HCS systems
Confocal scanning system
Research grade fluorescence scope
Basic clinical scope
Simple optical microscope
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• ??????????

Cell Lab Quanta
6 color plus cytometer / sorter high-end res.
5-6 color cytometer clinical / high-end res.
2-4 color cytometer simple assays
Automated Cell viability
Simple cell counters sizers
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CONFOCAL -SUB-CELL OR STRUCTURE ORIENTED IMAGING
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?????
(FLOW CYTOMETRY)
???????( flow cytometer ) ???????????????????
??(????????)????????? ?????????????????
??????? ??????? ????????
???????
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• ???????????
• 1)?????
• 2)????????????
• 3)?????????????-???????

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????????

• ??????
• ??????????
• ???????????Qdots

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Qdots-??????????
?????????????????
Qdots 605??????????(VERY SHARP)
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????????????????
?????????????? ?????????????
??????????
?????
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??????????????

• ???????
• ????????????????
• 4??????lt 1
• ????
• ???
• ????
• ?????????????
• 20?????,?????
• ???????
• ????

??????? VS ??????
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• ????????????!!!
• 1)??????
• 2)????????
• ???
• ?????
• 3)??????????,? PS ?ANNEXIN V???

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(No Transcript)
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????????????

• ???????-???????????
• FC???Fc????
• ????
• ?? IgG-CH3??????FcR??
• IgE-CH4??????FcR??

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??????????????

• ???????
• ????????????????
• 4??????lt 1
• ????
• ???
• ????
• ?????????????
• 20?????,?????
• ???????
• ????

??????? VS ??????
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T???????????????
???????
????
? ?
?? ??
4-8-
????
MHCII
MHCI
4-8-
?? ??
? ? ?
CD4 CD8-
CD4-CD8
CD4-CD8-
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CD?????????

• ??? CD??
• T?? CD2 CD3 CD4 CD5 CD7 CD8 CD28
  CD38
• B?? CD10 CD19 CD20-CD24 CD37
  CD72-CD75
• ???? CD13 CD14 CD15-CD17 CD33 CD34 CD35
• NK?? CD16 CD56 CD57 CD94
• ??? CD31 CD41 CD42a CD42b CD61 CD62p
  CD63
• ???? CD26 CD30 CD69 CD95-CD97
• ???? CD11a-c CD18 CD29 CD44 CD49a-f
• ???? CD105 CD106 CD109
• ?????? CD25 CD115 CD117 CD122 CD126

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• ??????????
• ??????,?????
• ??????
• ?????
• ???
• ????

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????

• ??
• ???
• ??SHEATH
• ???SAMPLE ?????51051106?/ml
• ??
• ??
• ??
• ?????PMT HV
• ??
• ????HV?GAIN ??
• A/D??
• ??
• ???

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??

• ?????
• ???????50-300 µm ??????,????? ???????
• ????,?????????

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• ------?????????????
• ????????????????

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?? - ????

• ???? ????????????
• ???? ????????????
• ???? ???????????
• ?? 45o ??????,?????????????,?????????????????
  ,???????????????( dichroic filter)

630 nm BandPass Filter
White Light Source
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????? ????????
FS
SS
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??

• ?????
• FS ????
• SS ?????
• ????
• FL1 FITC
• FL2 PE
• FL3 ECD
• FL4 PC5
• FL5 - PC7

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????
?? ???????(??????)??????????, ??????????????? ???
??????????,???????????????????
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????-??????????

• ?? volts
• ?? Gain

???????????????
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????

• Need to do when we measure signals from more
  than
• 1 fluorescence.

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4 Color Single Laser (488nm)FITC/PE/ECD/PC5
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??????????
??????1
??????2
??????3
??????4
??????5
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??
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Beckmancoulter -???????????
?????????????(???????????????????)
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???????

• ??????(PROTOCOL)
• ????(????,???,????)
• ????????
• ????,??????
• ????,????,?????????????

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(No Transcript)
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???????

• ???????????????
• ??????????????
• ???????????????
• ?????????(??????????????????)
• ?????????
• ????,???????
• ???????????????

• BECKMAN COULTER?????????????-Epics
  ALTRA????????????
• BD?FACSVantage????????JIA???,?????,?????,?????
• BD?FACSAria???????,??????????????,???????????????
  
• Cytomation?Moflow??????????,????????,?????????,???
  ????????,????

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????????????
FALS Sensor
488 nm laser
Fluorescence detector
-
Charged Plates
Single cells sorted into test tubes
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???????
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CELLS OF THE IMMUNE RESPONSE

• INNATE
• PMN NUETROPHILS
• EOSINOPHILS
• BASOPHILS
• MONOCYTES / MACROPHAGES
• DENDRITIC CELLS mDC - pDC
• NK CELLS
• NKT CELLS
• gd T LYMPHOCYTES
• CD5 B CELLS

• ADAPTIVE
• B CELLS
• HELPER T-CELLS
• Th1
• Th2
• REGULATORY T-CELLS
• (Th3)
• CYTOTOXIC T-CELLS

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????????

• ??????
• ???T????
• ?????????
• ???T??
• ?????
• ??????

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??????
T????(CD3) ? ? ? ?
CD3CD4 ???/???T??(Th/Ti) ??????????????
CD3CD4CD29 CD3CD4CD29- ???T??(Th) ???T??(Ti) ??????????? ???????????
CD3CD8 ???/???T??(Ts/Tc) ??????/??????
CD3CD8CD28- CD3CD4CD29- ???T??(Ts) ???T??(Tc) ????????? ????T??
CD3CD4CD8 ???T?? ?T?????????
CD3CD4-CD8- ??????T??,??? ?/? T????(TCR ?/? ) ??????T??,??? ?/? T????(TCR ?/? )
CD4/CD8??
CD45RACD45RO- ???/???T???? ??????????????????/????????????????????
CD45RA-CD45RO ???T???? ???????, ????????, ?HIV?????
CD45RACD45RO ???T??, ????? ?????
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??????
??T???? ? ? ? ?
CD3HLA-DR ??T??
CD4HLA-DR ??????/???T??
CD8HLA-DR ??????/???T??
CD4CD25 ??????/???T??
CD8 CD25
CD8CD38HLA-DR ??????/???T?? ??????????? ??????HIV???????
B????(19)
CD19CD23 ???B?? ???????
CD19CD5 ???????????
CD19CD5CD23 ??B?????????B????
NK??CD3CD(1656) ??????????, ????????
CD3CD57 CMV(?????)???????
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??T????????????????CD4?CD8??
?? CD4 T
h1CD4IFNr
Th2CD4IL4
Th0CD4IFNrIL4
??CD8
Tc1CD8IFNr
Tc2CD8IL4
Tc0CD8IFNrIL4
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Th1Th2??????????

• ??????????????????,??CD4T??????Th1?Th2
• Th1????IL 2?IL 12?IFN ??TNF ß?????,???????????????
  ??????,??????????????????????
• Th2????IL 4?IL 5?IL 6?IL 10?IL 13?????,??????????,
  ???????
• ???,CD8T???????Tc1?Tc2

• Th1?????????????T????????????????????????????????
  ?????????,??????????,????????????????????????
• Th2?? ??????,??B???????????,?IgG1?IgE????????,???
  ????????????,???IgE??????????????

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???????????????
?? CD3 CD4 CD8 CD4/CD8 NK
????? ?? ?? ?? ??
AIDS ?? ???? ?? ??
SARS ?? ?? ?? ?? ??
??????????? ?? ??? ?? ?? ??
?? ?? ?? ?? ?? ??
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?????T????

• ???(tetramer)??
• ????????
• ELISPOT
• Cytokine Secretion Assay

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TETRAMER

• ??????(APC)?????????TCR?????,?APC?T???MHC?????????
  ?????????T????????????MHC-??????????????T?????????
  ????????????????????T?????,???????????????????????
  ?????
• ?????????????????????????????????????????,??????
  ??????,???????????????????????????,??????????????
  ?????????????????T???????,?????????????????
• ?????MHC???????????????????????????MHC??a3???????
  ??(iTAg),?????????T????,????????????MHC?????????,?
  ???????????????,?????MHC??(iTAg)????,????????

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Beckman Coulter??????

• ???????
• HIV gag SLYNTVATL HLA - A0201
• HIV pol ILKEPVHGV HLA - A0201
• CMV PP65 NLVPMVATV HLA - A0201
• EBV GLCTLVAML HLA - A0201
• EBV GLCTLVAML HLA - A0201
• Influenza M1 GILGFVFTL HLA - A0201
• HBV core FLPSDFFPSV HLA - A0201
• ???????
• Mart1 ELAGIGILTV HLA - A0201
• Gp 100 IMDQVPFSV HLA - A0201
• Gp 100 ITDQVPFSV HLA - A0201
• Her-2/neu KIFGSLAFL HLA - A0201
• Her-2/neu RLLQETELV HLA - A0201
• PR-1 VLQELNVTV HLA - A0201
• Tyrosinase YMDGTMSQV HLA - A0201

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????????????iTopia Epitope Discovery System

• ???????????????????????????,iTopia????????????????
  ???????????????????????????

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(No Transcript)
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• ???????????????
• TH1????? ???????
• TH2????? ???????
• TH17????? ???????

PMA,IONOMYCIN,BFA?????????IFN-GAMMA
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• ?????CD8 T????

??HBV????(8AA)???CD8 T??
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(No Transcript)
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• ???????????
• TETRAMER????CD3,CD8???????????

???CD3,CD8???????,TER??
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• ????

TETRAMERCD8????
TETRAMERCD3????
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ELISPOT

• ELISPOT????????(???????????10-6,ELISPOT??????10-
  8)
• ELISPOT????????,????????????????????

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Cytokine Secretion Assay

• ??????

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CD4CD25 Treg

• ???T????????????????,???????,???????????
• ???T????????????????
• ???T??????CD4CD25bright,??????????????????????(eg
  .FOX-P3)
• CD25?????????????,??????????????

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???????(Dendritic Cells,DC)

• DC??????????,?????????????,????????????????,??????
  ??????
• DC??????????????????????,??????-PDC,MDC
• ??DC?????
• 1)Lin-,CD11c,CD123,??????CD80,CD86,CD83,DR?
• 2)??BDCA1,2,3,4?????????DC????
• 3)CD1416/CD85/CD33,CD1416/CD85/CD123

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3 contexts for DC DISCOVERY
Peripheral blood DC
FLOW CYTOMETRY
DC in tissues
DC generated in vitro
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???????-MDC/PDC
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??DC???-Regulatory DC

• Regulatory DC???DC??,?????,???T??
• ???????????,?????DC?????
• ?????????????,??DC???

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???????????

• dT??????T???10??,??????????,????????????????,??C
  D3????T???
• NK??????????8-25,?????????????NK??????NKR??????,
  ?????????????NK???????
• DC???????????,??????,????????????IFN-alpha?????

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???????
??????CCR5?HIV???
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?????

• ???????
• ?????
• ??????
• ?????
• ?????

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????-??????

• ????????????
• ??????????????????????????????
• ??????,??????
• ???????????????????
• ???????????,????,???????
• 1)?????CD102,CD56,CD54,CD166?,?CD62P
• 2)????????????CD121-126?,?CD25
• 3)??????CD49,CD29?,?CD11
• 4)?????CD105,CD90,CD73?,?CD34,CD45,CD80,CD86.

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• CD34 ISHAGE Single Plateform

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????-(??)???

• ????????????????????
• ??????????????????????
• ?????????,????????
• ??????,???????????HOECHEST 33342???????????
• ???????????????????????

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???????????-Hoechst 33342 side population

• ??????ABCG2???,?HOECHST 33342??????????,??????????
  ?????????????????????????,?????????????????????

QUANTA??SP??
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SIDE POPULATION????

• ???355nm???50mw?????
• 488BP and 488DL for SSC
• 608LP Hoechst-red
• 515SP Hoechst-blue
• ?????????-Goodell, M., et al. (1996) J Exp Med
  183, 1797-806

ALTRA??SP??
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???????????
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??????

• 1.???????Annexin ?/PI
• 2.?????Annexin ??Apo 27?TUNEL
• 3.????????Caspase?Fas?FasL
• bcl-2/bax
• 4.??????????

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?????????????
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DNA?????????
???????????(G0,G1,S,G2 ,M)?????,DNA???????????
??????????????DNA,???????????,???????????????,???G
0/G1,S?G2/M?????????????????????????????(CYCLIN
)?Ki67??????(PCNA)?,????????????G0?G1?S?G2?M.
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Normal Cell Cycle
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?????????

• CFSE??????
• Regulatory proteins (bax/bcl-2 family,
  p53)Forward and side scatterDye uptake
  (YO-PRO-1 PI)Membrane inversion (Annexin
  V)Loss of mitochondrial membrane potential
  (DiOC6)Caspase activation (Ab)DNA strand break
  (TUNEL, Sub G1)

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?????CD45???FS/SS??????
T-all
B-ALL
M6
M7
M4
M5
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M2
M3
CD45/SSC??????? CD45?????????,
CD45????????????,???????????????????????(?????????
????)??? CD45???????(SSC)????????,?????
SSC??,????????????? ????????????
CD45/SSC????????????????????????????????????????,?
??????????????,??????????,??????????50???????????
???
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????????BBA???????10?????
FL4(Differentiator Parameter)
Human Th1/Th2 Cytokine I Kit
FL2 (Reporter Parameter)
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Comparison of FlowCytomix and ELISA Standard
Ranges
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FlowCytomix Correlation with ELISAs
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Microsphere????

• ????????????,??????????
• ??
• ??4.4 ?5.5 µm ????????
• ???????????
• ???????????????????
• ELISA???.

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Kit Configurations

• Kit Types
• Simplex Kits
• Multiplex Kits (up to 10)
• Basic Kits
• Species/ Application
• Human Th1/Th2
• Human Cardio-Vascular
• Mouse Th1/Th2
• Rat Cytokine
• Human Adhesion

Main Menu
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Kit Configurations

• Simplex Kits
• Contain reagents for the detection of one
  individual cytokine
• User may combine Simplex kits for custom assay
• Basic Kit required
• Multiplex Kits
• Contain reagents for the detection of up to 10
  analytes per sample simultaneously
• Common Sample Types
• Serum
• Plasma
• Cell culture supernatants
• Whole Blood

Main Menu
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Product Characteristics
Main Menu
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Simplex Kits
Human Analytes
Mouse Analytes
IFN-g IL-8 sVCAM-1 IFN-g IL-5
IL-1b IL-10 t-PA GM-CSF IL-6
IL-2 IL-12p70 sP-selectin IL-1a IL-10
IL-4 TNF-a sCD40L IL-2 IL-17
IL-5 TNF-b MCP-1 IL-4 TNF-a
IL-6
Up to 10 Simplex Kits can be combined Except for
Simplex kits with identical bead populations
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FlowCytomix Multiplex Kits

• Human Th1/Th2 10 plex
• Th1/Th2 Cytokines Multiplex Kit I
• (IFN-g, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8,
  IL-10, TNF-a, TNF-b)
• Th1/Th2 Cytokines Multiplex Kit II
• (IFN-g, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8,
  IL-10, IL-12p70, TNF-a)
• Human Cardiovascular 7 plex
• (sCD40L, IL-6, IL-8, MCP-1, sP-selectin, t-PA,
  sVCAM-1)
• Mouse Th1/Th2 10plex
• (GM-CSF, IFN-g, IL-1a, IL-2, IL-4, IL-5, IL-6,
  IL-10, IL-17, TNF-a)
• Human Adhesion 6 Plex
• (sE-selectin, sICAM-1, sICAM-3, sPECAM-1,
  sP-selection, sVCAM-1)
• Rat Cytokine 6 plex
• (rGM-CSF, rIFN-g, rIL-1a, rIL-4, rMCP-1, rTNF-a)

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Cardiovascular 7-plex Details

• Human Cardiovascular 7-plex
• (sCD40L, IL-6, IL-8, MCP-1, sP-selectin, t-PA,
  sVCAM-1)
• These analytes are useful in cardiovascular
  research as well as the study of immune
  disorders.
• sCD40L a unique antibody used in the study of
  cardiovascular anomalies and immune disorders.
  Elevated levels have been found in research
  studies.
• sP-selectin - released by activated platelets and
  is an indicator of the number of platelets in
  blood.
• MCP-1 - induces mononuclear cell infiltration.
  Elevated levels have also been found in
  cardiovascular research studies.
• t-PA is used to study the development of
  cardiovascular abnormalities.
• VCAM-1 - elevated expression may be useful in
  cardiovascular research.
• IL-6 IL-8 used as a marker in the study of
  cardiovascular anomalies.

Main Menu
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Human Th1/Th2 Standard 10,000 pg/ml
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?????????????
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?????????

• ??DIL,DIO?????????????????

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???????????????????????? 1)???????????? 2)?????
???????????????? 3)????????????? 4)?????????????
????????? 5)?????????????????????? 6)???????????
??????? 7)????????????????????????,?T??(CD3,
CD4, CD8, gamma-delta TCR),NK??(CD3CD56),B??(CD3CD
19)? 8)?????????????????????????,????T, B,
NK??(??CD25, CD69, HLA-DR?) 9)??????????????,????
???(?????, DC),???T??(CD4CD25FOX-P3) 10)?????????
????????????(??Th1Th2?????)?
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????????????????

• ???????????????HLA?,??CXCR4,CCR5,DC-SIGN(?AIDS???
  ??),NKR
• ??????????,?????(???????,??????,???HOMING,?????,??
  ?????)
• ????(?????????????????????)
• ????????????????????????????????????????????
• ??????????????????
• ???????????????(?MPE,CONFOCAL,IC200?)