fast, accurate apoptosis and cell cycle studies … · fast, accurate apoptosis and cell cycle...
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BD Confidential: Internal use only
Fast, Accurate Apoptosis and Cell Cycle Studies Using Kits and Templates on the BD Accuri™ C6Presented by:s, PhD, Marketing Applications Specialist, BD BiosciencesStacey Roys, Scientist, BD BiosciencesLora Tirri, BD Accuri Product Manager, BD Biosciences
23-16098-00
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Outline• Background: Apoptosis and Cell Cycle• Introduction to the BD Accuri™ C6 Flow Cytometer• BD Kits and Templates• BD Templates on the Web• How to Create Your Own Templates• Coming Soon….
BD Confidential slide 2
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Outline• Background: Apoptosis and Cell Cycle• Introduction to the BD Accuri™ C6 Flow Cytometer• BD Kits and Templates• BD Templates on the Web• How to Create Your Own Templates• Coming Soon….
BD Confidential slide 3
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Apoptosis• Definition:
– The process leading to controlled self destruction of a cell. Cells undergo death neatly without damaging their neighbors. Apoptosis is a “programmed event.”
• Importance of Apoptosis– Cell termination
• Viral infection, cancer– Homeostasis
• Tumor, diseases – Development
• Organs, appendages, patterning– Lymphocyte development
• Thymic selection– Drug discovery studies
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Apoptosis at a Glance
1. Phosphotidyl serine (PS) exposed
2. Mitochondrial potential decreases
3. Caspases activated
4. DNA fragmentation
3
4
2
1Apoptosis induction
Apoptotic cell
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Summary of Apoptosis AssaysFeature Measured Assay Key Benefits
Plasma Membrane Alterations
Annexin V Binding Assay:• Single Conjugates• Annexin V Kits
•Detects early apoptosis•Quick and easy
Mitochondrial Changes • BD™ MitoScreen •Quick and easy
Caspase Activation • Active Caspase-3 Flow Kit •Specific antibodies can detect activated vs uncleaved caspase-3•Can be multiplexed
DNA Fragmentation • APO-BrdU TUNEL Assay• APO-Direct TUNEL Assay
•Works with adherent cells
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Apoptosis: Scatter Properties
Formation of apoptotic vesicles •Increases side scatter
Reduced refractive index of apoptotic cells •Decreases forward scatter
Cell shrinkage during apoptosis is associated with a decrease in forward scatter. Analysis of light scatter often is combined with other assays.
Untreated Camptothecin treated
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Apoptosis Method Decision TreeCells
Flow
Unfixed Fixed
Loss of membrane symmetry: Annexin V*
Mitochondrial membrane potential: JC1*
Caspase inhibitors addition: VAD-FMK*
Cleaved Markers: Active Caspase 3*, PARP*
DNA fragmentation: TUNEL assay/APO-BrdU kit*
* BD products available
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Cell Cycle and Proliferation Analysis• There are several methods available to
measure cell cycle and proliferation.
• Flow cytometry allows quantitative detection of results.
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Cell Cycle Analysis of a Population Stained for Incorporated BrdU and Total DNA Levels (7‐AAD)
BrdU
‐FITC
BrdU
‐APC
7 AAD 7 AAD
Sub‐G0/G1
S phaseS phase
Human peripheral blood mononuclear cells (PBMCs) were stimulated with anti‐CD3/CD28 for 48 hours and re‐stimulated with PMA+Ionomycin for 4 hours, and BrdU was added for the final 1 hour. Cells were then harvested and stained using the BrdU staining protocol.
G2+M phase
G0/G1
Sub‐G0/G1
G2+M phase
G0/G1
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Outline• Background: Apoptosis • Introduction to the BD Accuri™ C6 Flow Cytometer• BD kits and templates• BD templates on the web• How to create your own templates• Coming soon….
BD Confidential slide 11
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IntroductionThe BD Accuri™ C6 Flow Cytometer System
An Affordable, Full-Featured, Easy-to-Use Flow CytometerTwo lasers and six detectors
BD Confidential slide 12
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Intuitive Software
Sample Grid
CytometerStatus
Fluidics Controls
Run Criteria
Real-Time Updates
BD Confidential slide 13
Histograms
Dot Plots
Density Plots
Analysis and Gating Tools
Plot Statistics
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Outline• Background: Immunophenotyping • Introduction to the BD Accuri™ C6 Flow Cytometer• BD Kits and Templates• BD Templates on the Web• How to Create Your Own Templates• Coming Soon….
BD Confidential slide 14
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Arguably, as easy as cell analysis is going to get.
BD Kits and Templates
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BD Cell Biology Kits
BD Confidential slide 16
Application Kit Cat. No.
Apoptosis Annexin V FITC Apoptosis Detection Kit II 556570
Annexin V PE Apoptosis Detection Kit I 559763
MitoScreen (JC-1) Kit 551302
Caspase-3 PE Assay Kit 550914
Cell Cycle and DNA DNA Reagent Kit 340242
FITC BrdU Flow Kit 559619
APC BrdU Flow Kit 552598
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A BD Accuri™ template is a pre-defined workspace that includes gates, labels, run criteria, and compensation
settings for a specific assay.
A BD Accuri™ template is a pre-defined workspace that includes gates, labels, run criteria, and compensation
settings for a specific assay.
What is a Template?
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Pre‐Defined Gating and Run Criteria for Ease of Use
BD Confidential slide 18
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Compensation
BD Confidential slide 19
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Where to Find the Templates• www.bdbiosciences.com/go/templates
BD Confidential – 20
Kits and Templates for the BD Accuri™ C6Category Product Information Sheet Brand Kit Cat. No. Template
Cell Biology
BD Apoptosis Kits and Templates
BD Pharmingen™ Annexin V FITC Apoptosis Detection Kit II 556570 Download
BD Pharmingen™ Annexin V PE Apoptosis Detection Kit I 559763 Download
BD™ MitoScreen (JC‐1) Kit 551302 Download
BD Pharmingen™ Caspase‐3 PE Assay Kit 550914 Download
BD Cell Cycle and DNA Kits and Templates
BD Cycletest™ Plus DNA Reagent Kit 340242 Download
BD Pharmingen™ FITC BrdU Flow Kit 559619 Download
BD Pharmingen™ APC BrdU Flow Kit 552598 Download
Immunology
BD Naïve/Memory T‐Cell Kits and Templates
BD Multitest™ CD45RA/CD45RO/CD3/CD4 340571 Download
BD Multitest™ CD45RA/CD62L/CD3/CD4 340977 Download
BD Pharmingen™ Human Naïve/Memory T Cell Panel 561438 Download
BD Intracellular T‐Cell Kits and templates
BD Pharmingen™ Th1/Th2/Th17 Phenotyping Kit 560751 Download
BD FastImmune™ IFN‐γ/CD69/CD8/CD3 Kit 346048 Download
BD FastImmune™ IFN‐γ/IL4 Kit 340456 Download
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How to Open a Template
BD Confidential slide 21
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How to Open a Template
BD Confidential slide 22
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How to Use a Template: Example Using the Annexin V Apoptosis Detection Kit (Cat. No. 556570)
BD Confidential slide 23
Reagents:Annexin V FITCPropidium Iodide
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How to Use a Template: Verify Settings, Collect, Adjust as Necessary
BD Confidential slide 24
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Ready to Acquire Data
BD Confidential slide 25
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Annexin V FITC Data
BD Confidential slide 26
DMSO Camptothecin
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How to Further Optimize a Template: BD Cycletest™ Plus DNA Reagent Kit (Cat. No. 340202)
BD Confidential slide 27
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How to Optimize a Template: Zoom Tool
BD Confidential slide 28
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How to Optimize a Template: Statistics Tab
BD Confidential slide 29
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BD Cycletest Plus Data
BD Confidential slide 30
PBMC MFI=403,211
K562 MFI=580,150
DNA Index= 1.4
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How to Optimize a Template for the BD CSampler™ Accessory: BD Pharmingen™ Active Caspase‐3 PE (Cat. No. 550914)
BD Confidential slide 31
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BD Confidential slide 32
How to Optimize a Template: BD CSampler
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How to Optimize a Template: BD CSampler
BD Confidential slide 33
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How to Optimize a Template: BD CSampler
BD Confidential slide 34
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BD Pharmingen™ Active Caspase‐3 PE Kit Data
BD Confidential slide 35
DMSO Camptothecin
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Application‐specific Templates: BD MitoScreen (Cat. No. 551302)
BD Confidential slide 36
Untreated CCCP
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Application‐specific Templates: BD Pharmingen™FITC or APC BrdU Flow Kits (Cat. No. 559619 or 552598)
BD Confidential slide 37
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Outline• Background: Apoptosis• Introduction to the BD Accuri™ C6 Flow Cytometer• BD Kits and Templates• BD Templates on the Web• How to Create Your Own Templates• Coming Soon….
BD Confidential slide 38
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6,8-Peak BeadsBD™ CBAWater Quality
Cell Cycle/Proliferation: BrdU Apoptosis: Annexin V Apoptosis: Caspase-3DNA AnalysisBD MitoScreen Stem Cell Flow KitsT-Cell CytokinesNaïve/Memory T Cells
BD Templates Available for Free on the Web
BD Confidential – 39
EXISTING
NEW
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Outline• Background: Apoptosis• Introduction to the BD Accuri™ C6 Flow Cytometer• BD Kits and Templates• BD Templates on the Web• How to Create Your Own Templates• Coming Soon….
BD Confidential slide 40
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How to Create Your Own Templates
BD Confidential slide 41
1. Open a new workspace or existing workspace.
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How to Create Your Own Templates
BD Confidential slide 42BD Confidential slide 42
2. Collect samples, optimize plots and run criteria to desired settings.
3. If desired, input compensation, parameter names, and threshold settings and click Apply to:All samples.
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How to Create Your Own Templates
BD Confidential slide 43BD Confidential slide 43
4. Select File > Save Template As.
5. Save the template with a new name.
Note: Creating templates works bestat the cytometer workstation, when theBD Accuri C6 is powered on.
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Important Points to Remember• Customize templates to fit your sample type and
workflow.• Verify all settings, including thresholds and
compensation.• When using the BD CSampler, copy desired settings to
the Auto Collect Tab.• For best results, create templates when the software is
connected to the cytometer.
BD Confidential slide 44
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Outline• Background: Apoptosis • Introduction to the BD Accuri™ C6 Flow Cytometer• BD Kits and Templates• BD Templates on the Web• How to Create Your Own Templates• Coming Soon….
BD Confidential slide 45
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6,8-Peak BeadsBD CBAWater Quality
Cell Cycle/Proliferation: BrdU Apoptosis: Annexin V Apoptosis: Caspase-3DNA AnalysisBD MitoScreen Stem Cell Flow KitsT-Cell CytokinesNaïve/Memory T Cells
TregsMouse LymphocytesRat LymphocytesMouse TregsMouse T-Cell CytokinesCounting and Viability of BacteriaMesenchymal Stem Cell Kit
BD Templates Coming Soon to the Web…..
BD Confidential – 46
EXISTING
NEW
COMING SOON
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Special Promotional OfferFor a limited time, take advantage of big savings and value-added packages for the
BD Accuri™ C6 flow cytometer with our reagent kit discount.
Get a 40% discount on select reagent kits now through June 2014.
New to BD Accuri?
Buy a BD Accuri C6 now and receive an immediate 10% discount on the purchase price. You'll also be eligible to receive
40% off the list price of all BD Pharmingen™ reagentsyou purchase for use on the BD Accuri C6 for the next 2 years.
Offer expires June 30, 2014.
Now you can simplify your workflow while dramatically reducing the cost of use of your personal flow cytometer.
www.bdbiosciences.com/go/templates
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Summary
BD Confidential slide 48
• The BD Accuri™ C6 is making it even easier to apply the power of flow cytometry to your research with free software templates and ready-to-go reagent kits specific to your studies.
• Find out more about how the BD Accuri C6 puts the power of 4-color cell analysis within reach by visiting www.bdbiosciences.com/go/templates
+ + =Flow Cytometry Within Reach.™
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