allcells flow cytometry webinar 09-26-2013
DESCRIPTION
The biotechnology and pharmaceutical industry is continuing to implement more flow cytometry-based assays in research and drug discovery due to the flexibility in assay development. The variety of assays which can be performed continues to expand, giving research laboratories options in data collection ranging from detection of cell signaling pathway activation to immunophenotyping. This webinar will provide an introduction to flow cytometry, a technique that uses fluorescently conjugated antibodies and dyes to identify characteristics in heterogeneous populations of cells. In addition we will present case studies on how flow cytometry can be used in drug discovery.TRANSCRIPT
Your Reliable Source of Human Primary Cells
Flow Cytometry 101:How Flow Cytometry Can Help Advance Your Research
Components of Flow Cytometry
• Fluidics• Lasers• Optics• Data Analysis• Fluorescence- activated Cell Sorting
Fluidics System Transports Cells
Laser
Hydrodynamic Focusing
SheathFluid
LightScatter
Fluorescent Signal
Sample
Sample Flow Rate Impacts Alignment
High Sample Flow Rate =Small difference = Larger Core
Low Sample Flow Rate=Large difference = Smaller Core
Lasers are ideal for flow cytometry
http://notproperlydone.com
Light Amplification byStimulatedEmission ofRadiation
1. Stable2. Focused3. Single Wavelength
Lasers Excite Fluorophores
http://www.piercenet.comhttp://www.bdbiosciences.com
Optics Detect Emission of Fluorescence
http://medicine.umich.edu
•Dichroic Mirrors•Filters•Detectors
Multiparametric analysis is possible
http://www.bdbiosciences.com
Sample Preparation
• Incubate with Antibody• Wash to remove unbound Antibody • Analyze
Fluorescence-Activated Cell Sorting
Laser
Hydrodynamic Focusing
SheathFluid
LightScatter
Fluorescent Signal
Sample
++
+
--
--
waste
How can I use Flow Cytometry?
• Immunophenotyping• Intracellular proteins
• Transcription Factors• Phosphorylation Status
• Extracellular proteins• Receptor Density
• Viability/Apoptosis• Caspase activation• Annexin-V presentation• TUNEL
• Proliferation• CFSE• Cell Cycle Analysis
• Isolation/Sorting
Case Study – Immunophenotyping
Request:Characterize AML samples for expression of specific markers so customer could purchase samples of interest
Approach:Multiparametric analysis of CD47, CD147, CD9 and proprietary protein
Results:Identified specific patient samples that could be used in downstream analysis for customer
Case Study – Pathway Activation
Request:Treat MM samples with proprietary compound and determine phosphorylation status of protein
Approach:After treatment, fix and permeabilize cells and quantify phosphorylation of protein in cell subpopulations
Results:Compound inhibited phosphorylation of proteins associated with proliferation/ apoptosis in CD38- and CD138-expressing cells
CD38 CD1380
1000
2000
3000
4000 UntreatedTreated
Mea
n Fl
uore
scen
t In-
tens
ity
Case Study – Apoptosis
Request:Treat CLL samples with proprietary compound and determine apoptotic affect
Approach:After treatment, quantify apoptosis and necrosis using Annexin-V and 7-AAD
Results:Compound induced apoptosis/necrosis in CLL samples
Case Study – Receptor Density
Request:Determine receptor density of specific antigens in AML patients
Approach:Measure Mean Fluorescent Intensity (MFI) of antibodies compared with beads bound with absolute levels of IgG
Results:Quantified antigen binding capacity in multiple AML samples
Case Study – Multiparametric Sorting
Request:Isolate CD14+CD16+ and CD14+CD16- subpopulations from patient with RA undergoing treatment with proprietary compound
Approach:Samples sent from doctor at regular intervals were sorted based on CD14- and CD16-expression for RNA extraction and transcriptional analysis.
Results:CD14+CD16+ and CD14+CD16- fractions were isolated from fresh PB MNC.
Case Study –Sorting based on MFI
Request:Sort CD56-bright and CD56-dim populations for downstream analysis
Approach:Isolate CD56+ cells using immunomagnetic isolation and sort CD56 subpopulations based on MFI
Results:Isolated CD56-expressing cells based on relative expression level for RNA analysis
Flow Cytometry is a powerful research tool
Cell Isolation
Proliferation
Apoptosis (Annexin V/PI)Pathway Activation
Basha Stankovich, Ph.D.
Bioservices Manager
www.allcells.com/technical-support/
www.allcells.com/support/webinars/
Contact Information
Please use the prompt on the right of your screen to send a question to the organizer
Questions will be read and answered
Any offline questions may be sent to [email protected]
Thank you!
Q&A