Control - BD高通量活細胞共聚焦分析系統介紹.pdf

45 Cell-Bio 尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司

www.cell-bio.com.tw

[email protected] 02-27855860 0953062485

3.Mophometric Measurement 3.Mophometric Measurement

Make measurements on cells using structural dyes/antibodies:

Angiogenesis Angiogenesis

•• Used to determine the +/Used to determine the +/-- response of primary endothelial cells to compounds response of primary endothelial cells to compounds –– Tumor formationTumor formation

–– Wound healingWound healing –

– RetinopathyRetinopathy

–– Macular degenerationMacular degeneration

•• Model systemModel system –

– HUVEC-HUVEC-2 cells2 cells

–– MatrigelMatrigel Matrix coated platesMatrix coated plates

–– Network of tubules formed –Network of tubules formed – live cell stain & imaginglive cell stain & imaging

–– SuraminSuramintreatment –treatment – retards the formation of blood vesselsretards the formation of blood vessels

3.Mophometric Measurement 3.Mophometric Measurement

Make measurements on cells using structural dyes/antibodies:

Angiogenesis Angiogenesis

HUVEC angiogenesis on BD Matrigel^TM Matrix

47 Cell-Bio 尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司

www.cell-bio.com.tw

[email protected] 02-27855860 0953062485

3.Mophometric Measurement 3.Mophometric Measurement

Make measurements on cells using structural dyes/antibodies:

Angiogenesis: Quantitative Data Angiogenesis: Quantitative Data

Tube Nodes Points Tube Nodes Points Tube Count

Tube Count

Tube Segment Count Tube Segment Count Tube Branch Count Tube Branch Count Tube Max Length Tube Max Length

Tube Average Length Tube Average Length Tube Total Length Tube Total Length

1.0? 0^-7 1.0? 0^-6 1.0? 0^-5 1.0? 0^-4 1.0? 0^-3 0

10000 20000 30000 40000 50000 60000 70000

Log [M] Suramin Total Tube Area (pixels)

1.0? 0^-7 1.0? 0^-6 1.0? 0^-5 1.0? 0^-4 1.0? 0^-3 0

1000 2000 3000 4000 5000 6000 7000 8000

Log [M] Suramin Total Tube Length (pixels)

1.0? 0^-7 1.0? 0^-6 1.0? 0^-5 1.0? 0^-4 1.0? 0^-3 0

50 100 150 200 250 300 350

Log [M] Suramin Tube Complexity (total # of segments)

3.Mophometric Measurement 3.Mophometric Measurement

Make measurements on cells using structural dyes/antibodies:

Angiogenesis:

Angiogenesis: Neurite Neurite Outgrowth Outgrowth

PC-12 cells induced with NGF

•Nuclear Mask:

Cell Segment

•Nurite Mask:

Nurite Segment

•Merge:

49 Cell-Bio 尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司

www.cell-bio.com.tw

[email protected] 02-27855860 0953062485

3.Mophometric Measurement 3.Mophometric Measurement

Make measurements on cells using structural dyes/antibodies:

Neurite

Neurite Outgrowth Outgrowth

3.Mophometric Measurement 3.Mophometric Measurement

Make measurements on cells using structural dyes/antibodies:

Miotube

Miotube Fusion Fusion

DMSO 52.7 uM Nifedipine 267 uM Nifedipine

51 Cell-Bio 尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司

www.cell-bio.com.tw

[email protected] 02-27855860 0953062485

3.Mophometric Measurement 3.Mophometric Measurement

Make measurements on cells using structural dyes/antibodies:

Miotube

Miotube Fusion Fusion

Number of Nuclei in Myotubes

0 1000 2000 3000 4000 5000 6000 7000 8000

1.6E-05 2.3E-05

3.5E-05 5.3E-05

7.9E-05 1.2E-04

1.8E-04 2.7E-04

4.0E-04

uM Nifedipine

# Nuclei

Total # Nuclei

# Nuclei in Myotubes

This confocal image is using BD 96-Well Optilux Microplates to increase image

quality

4.Cell Movement 4.Cell Movement

• • Measure cellular mobility/motility and invasion Measure cellular mobility/motility and invasion Invasion

Invasion

DMEM + 5% FBS

DS Red - Migrated Sytox Green - Migrated Hoechst - Migrated BSA Control - Migrated

BD Falcon™ HTS FluoroBlok™

96-Multiwell Insert System

53 Cell-Bio 尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司

www.cell-bio.com.tw

[email protected] 02-27855860 0953062485

4.Cell Movement 4.Cell Movement

• • Measure cellular mobility/motility and invasion Measure cellular mobility/motility and invasion Wound Healing Assay

Wound Healing Assay

4.Cell Movement 4.Cell Movement

• • Measure cellular mobility/motility and invasion Measure cellular mobility/motility and invasion Wound Healing Assay

Wound Healing Assay

Image analysis and record by

BD IPLab BD IPLab V4 Software V4 Software

55 Cell-Bio 尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司

www.cell-bio.com.tw

[email protected] 02-27855860 0953062485

•Fly embryo •Fly embryo gastrulationgastrulation

•

•TubulinTubulin. .

•PlanApo•PlanApo 60x 1.4 NA60x 1.4 NA

•

•0.5um steps0.5um steps

•xz•xz, , yzyz (1(1--256)256)

•George von •George von DassowDassow

•Friday Harbor, UW•Friday Harbor, UW

3D Reconstruction

Image Acquire by

Image Acquire by BD Pathway BD Pathway

855 855 with confocal with confocal module module

3D Reconstruction

Image Acquire by

Image Acquire by BD Pathway 855 BD Pathway 855 with confocal with confocal module module

57 Cell-Bio 尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司

www.cell-bio.com.tw

[email protected] 02-27855860 0953062485

Time Lapse

Image Acquire by Image Acquire by BD Pathway 855 BD Pathway 855

with environment control with environment control

••Cell divisionCell division

•

•Sand dollar embryosSand dollar embryos

••Injected with rhodamineInjected with rhodamine--tubulintubulin

& AlexaAlexa 488 phalloidin488 phalloidin. .

•

•Early-Early- mid blastulamid blastula

••PlanApoPlanApo60x water lens60x water lens

••George von DassowGeorge von Dassow & Bill & Bill Bement

Bement

••Friday Harbor, UWFriday Harbor, UW

• • Dictyostelium Dictyostelium Feeding on Yeast Feeding on Yeast

• • Dictyostelium Dictyostelium – – GFP- GFP - cell cell membrane

membrane

• • 150ms, 13z steps, 30min 150ms, 13z steps, 30min

• • Cascade 512B Cascade 512B

• • David Knecht David Knecht Department of Department of Molecular and Cell Biology Molecular and Cell Biology University of Connecticut University of Connecticut

4D Image

Image Acquire by

Image Acquire by BD Pathway 855 BD Pathway 855 with environment control with environment control

59 Cell-Bio 尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司

www.cell-bio.com.tw

[email protected] 02-27855860 0953062485

Summary and Conclusions Summary and Conclusions

• • High content analysis provides a unique High content analysis provides a unique opportunity to investigate spatial and

opportunity to investigate spatial and temporal events in living cells

temporal events in living cells

• • Kinetic measurements can provide useful Kinetic measurements can provide useful information (rates of change)

information (rates of change)

• • Managing kinetic data requires real Managing kinetic data requires real - - time data time data acquisition and data reduction capabilities

acquisition and data reduction capabilities

• • Multiplexed kinetic experimentation requires Multiplexed kinetic experimentation requires biological events to be co

biological events to be co- -occurring occurring

Any Question Any Question

Thanks For Your Patient & Attention!!

Ce ll-B io

尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司尚博生物科技有限公司

www.cell-bio.com.tw

Glenn Yang Glenn Yang

[email protected]

Dalam dokumen BD高通量活細胞共聚焦分析系統介紹.pdf (Halaman 44-60)