Flow Cytometry Core A Resource of the Section of Immunobiology, Department of Internal Medicine, and the YCCC.
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Flow Cytometry Core A Resource of the Section of Immunobiology, Department of Internal Medicine, and the YCCC Presentation Overview • Flow cytometry background and technical review • Yale Flow Core: history, capabilities, people, and operation. • Use of flow cytometry to study memory B cells in my own lab. What is Flow Cytometry? • The analysis of single cells, particles, or other discrete elements as they flow past one or more focused light sources based on reflected, scattered, or fluorescent light generated by those light sources. Flow Cell Injector Tip Fluorescence signals Focused laser beam Sheath fluid Forward Angle Light Scatter Laser FALS Sensor 90 Degree Light Scatter Laser FALS Sensor 90LS Sensor Fluorescence Detectors Laser Freq FALS Sensor Fluorescence Fluorescence detector (PMT3, PMT4 etc.) Fluorescence Activated Cell Sorting 488 nm laser FALS Sensor Fluorescence detector Charged PlatesAttract and focus droplets of opposite charge Single droplets sorted into test tubes - Computer analysis of detector data- “gating” leads to + or - charging of droplet + Optical Filters Dichroic Filter/Mirror at 45 deg Light Source Transmitted Light Reflected light Standard Band Pass Filters 630 nm BandPass Filter White Light Source Transmitted Light 620 -640 nm Light Standard Long Pass Filters 520 nm Long Pass Filter Light Source Transmitted Light >520 nm Light Standard Short Pass Filters Light Source 575 nm Short Pass Filter Transmitted Light <575 nm Light Flow Cytometry Optics PMT 4 Flow cell PMT Dichroic Filters 3 PMT 2 Bandpass Filters PMT 1 Laser What can flow be used for? • Expression of cell surface or intracellular proteins or neo-epitopes such as phospho-proteins generated via cell signaling (after permeabilization) using fluorescently tagged Abs. • Use of small molecule, protein, or particle-based probes to detect: Ca++ flux, pH, mitochondrial polarization (apoptosis surrogate), caspase activation, cell division via dye dilution (e.g. CFSE), phagocytosis, DNA content (cell cycle analysis) or proliferation (BrdU incorporation). • Detection of intrinsic cell fluorescence based on expression of fluorescent proteins from reporter constructs. • Analysis of cell size and complexity using light scatter. • Multiparameter analysis to determine cellular heterogeneity and to link properties to cell phenotypes in complex mixtures. • Quantitative technique to enumerate specific cell types. • Preparative method that can combine any of the above techniques isolate cells or even subcellular fractions at a rate of up to 50K events/second. The Yale Flow Facility History • Began as 1 FACScan and FACStar plus, supported by HHMI and Section of Immunobiology. • FACScalibur added in 94. • FACSVantage in 98. • FACS-DIVA upgrade in 2001-provided gratis by BD. • Winter 2002: Current core opened in TAC with space and instruments contributed by Internal Medicine and purchase of additional used MoFlo by YSM. Custombuilt rooms. • Initial configuration: 1 FACScan, 4 FACSCaliburs (3 from Int. Med), 2 sorters. • Shared Instrument Grant for FACSAria in 2003. • Fall of 2004: Purchase of LSRII-with UV capability for DNA analysis. • November 2004: YCCC merger completed. Instrumentation • FACS Analysis: – 3 Color analysis: FACScan – 4 Color analysis: 4 FACScaliburs – 11-Color analysis: LSRII (new) • Sorters: FACSVantage DIVA: hi-speed digital stream in air sorter with UV, 488, 633; 4-way sort; cloning; aerosol containment. MoFlo: hi-speed digital stream in air sorter with 488, 633; cloning FACSAria: hi-speed digital cuvette sorter with 407, 488, 633; hi sensitivity, 13-color; 4-way sort; cloning; aerosol containment TAC Building Room S617 7’-10” 7’-10” Filing Cabinet 2’ W x 5’ D FACS Aria/Computer 8’-9”W x 4’-5”D MoFlo 3’ D x 6’ W FACS Vantage 4’ D x 3’ W 2’ W x 2’D 3’ W x 3’ D 4’-7” 2’-6” 2’-6” 10’ 31’ Scale 0.3”-1’ - Bench Space 10’ 15’ 15’ Desk 4’-7”W x 2’-6”D 2’ 2’ 2’ 2’ 2’ 7’-10” Lasers and Colors Instr uments BD FACScan Laser Argon (L1) BD FACSCal ibur Argon (L1) BD FACSVantage Red Diode (L2) Argon (L1) HeNe (L2) BD FACSAr ia Argon (L1) HeNe (L2) BD LSR I I Violet (L3) Argon (L1) HeNe (L2) UV (L3) Cytomation MoFlo Violet (L4) Argon (L1) HeNe (L2) Exci tation Laser Line (nm) Fluorescence Channel 488 FL1 Green FL2 Yell ow FL3 Red 488 FL1 Green FL2 Yell ow FL3 Red 635 FL4 Red 488 FL1 Green FL2 Yell ow FL3 Red FL6 UV 633 FL4 Red FL5 Infr aRed 488 Green Yel low Red Far Red Infra Red 633 Red Infra Red 407 Infra Red 488 Green Yel low Red Far Red Infra Red 633 FL4 Red Infra Red 355 Violet Blue 407 Blue 488 Green Yel low Red Far Red Infra Red 633 FL4 Red Infra Red Fluorochromes FITC PE PE-Texas Red FITC PE PE-Texas Red APC FITC PE PE-Texas Red Hoechst APC APC-Cy7 FITC PE PE-Texas Red Per CP-Cy5. 5 PE-Cy7 APC APC-Cy7 Alexa Fl uor 405 FITC PE PE-Texas Red Per CP-Cy5. 5 PE-Cy7 APC APC-Cy7 Alexa Fl uor 405 Alexa Fl uor 405 FITC PE PE-Texas Red Per CP-Cy5. 5 PE-Cy7 APC APC-Cy7 Alexa Fl uor 488 PI PE-Cy5 Alexa Fl uor 488 PE-Cy5 Alexa Fl uor 647 Alexa Fl uor 488 PI PE-Cy5 Alexa 350 Alexa Fl uor 647 EMA Per CP Per CP-Cy5. 5 PE-Cy7 Per CP Per CP-Cy5. 5 PE-Cy7 EMA Per CP Indo-1 Per CP-Cy5. 5 PE-Cy7 Per CP PI EMA Per CP PI EMA Alexa Fl uor 488 PE-Cy5 Alexa Fl uor 647 Paci fic Bl ue Alexa Fl uor 488 PE-Cy5 Alexa Fl uor 647 Paci fic Bl ue Paci fic Bl ue Alexa Fl uor 488 PI PE-Cy5 Alexa Fl uor 647 EMA Per CP Capabilities and Techniques • User operated 3, 4, and 11-color analysis. • Technician-assisted analysis on request for an additional fee. • Multicolor hi-speed digital sorting: – – – – – 4-way Cloning/single cell or multicell Sterile UV, 407, 488, and 633 laser lines Operator performs most sorts; user-operation an option for experienced FACS Aria users. • Techniques: cell surface markers, cytokines, intracellular staining, live-dead discrimination, Ca++-signaling, DNA/cell cycle analysis, FRET, subcellular fractions, detection of almost any fluorescent molecule. • Assistance with data analysis: two workstations with appropriate software. Personnel • Mark Shlomchik has run facility for the last 7 yrs. Currently my 5% effort is supported by a PPG on which I am one of the PIs. • Two sorter operators paid by effectively 50% by HHMI: Tom Taylor and Gouzel Tokmoulina. • One R+D tech who supervises FACS Aria and analyzers: Geoff Lyon. • One new R+D tech who oversees the analyzers, billing and training: Don Foster. User Support and Resources • Consultation on sorts, analysis, etc. before, during or after the experiment, as needed, no charge. • Regular training sessions for all instruments. • Broadcast announcements via email about new policies, unexpected downtime, etc. • Sponsorship of training seminars on data analysis, new reagents, techniques. • Negotiated discounts with a variety of vendors. User Support and Resources • New web site: designed and maintained by Gouzel Tokmoulina. – – – – Description of equipment Facility rules Sign-up Resources and information • Web-based scheduling for all analyzers. Usage Statistics • • • • Analysis: ~950 hr./month Sorting: ~325 hr./month 119 Active laboratories >486 Registered individual users Budget • Sources of income – User fees: ~$440K – YCCC: $39K – HHMI: ~$112K • Costs – Salaries: ~$300K/yr. Salary+fringe for 4 people and administrative support. – Maintenance contracts: $135K/yr. (some deferred due to prepaid contracts. – Other: ~$25K/yr. Breakdown of Key Fees • User-operated analysis: $14/hr. • User-operated LSRII or Aria (analysis only): $22/hr. • Operator-performed sorting or analysis: $68/hr. • Training: $30/hr. for up to three people (1 hr. sessions). Future Plans • Additional LSRII in one to two years. We have applied for a Shared Instrument Grant that is pending review. • Further application development and customer support and training. • Expansion with a satellite facility in the Amistad Building to support Stem Cell, Vascular Biology and Human Immunology programs: will need a new sorter and an LSRII analyzer. All users could use either facility. • Expanded education mission. Applications Examples-Study of B cell Memory • Identifying memory cells and phenotyping them using BrdU labeling. • Sorting memory cells for mRNA or subsequent functional analysis. • Using FACS to confirm the microarray data. Definition of a “Memory Cell” • A cell that has previously responded to antigen and that persists in a resting state for a long period of time after initial exposure. • What are the features that distinguish resting “memory” and naïve B cells? Ig Transgenic mice with an increased Membrane IgM (mIgM) frequency of hapten NIP-specific B Transgene Construct cells s VDJ C E M switc h Bl/6, V H186. 2 Balb/c IgM a, nosecretedexo n Signals for production of secreted IgM have been deleted. Only membrane-bound IgMa is produced by the transgene. Pairing of the VH186.2 variable region gene with endogenous 1 light chain produces antibody specific for the hapten NP. This provides a system in which the functions of B cells and antibody can be distinguished. Expansion of Antigen-specific Population 12 weeks after Immunization Summary of NP+ Expansion after Immunization BrdU Labeling Strategy BrdU is administered intraperitoneally every 12 hours on the days indicated above. BrdU-positive Ag-specific B cells present in mIg and (m+s)Ig mice 12 weeks post immunization Decay Kinetics of BrdU-labeled Memory B cells is Equivalent in mIg and (m+s)Ig mice n=5-14 mice BrdU+NP+ Half life >8wks BrdU-positive, NP+ B cells present in mIg and (m+s)Ig Immune mice were compared with NP+ cells in Alum control mice Resting Memory Cells Have High CD80 Expression Bold=immune A. A System for Generating Large Numbers of Memory Cells for Further Study: Hyperimmunization of mIg mice NP-CGG - 4wk B. NP-CGG d0 Assay C. 8 12 16 20wk Naive Two Doses 7.96 B220+ 1.66 NP+ 12 weeks post 2nd immunization APPROACH 93.8 Naïve or Immunized a-NP Splenocytes Naive FACS Sorting NIP RNA Isolation 1.68 Memory 92.4 Labeled cRNA Preparation 3.16 Affymetrix Hybridization Statistical Analysis Data Mining Kappa Biological Replicates #1 Naïve NP 3 Memory NP 4 GC NP #2 4 4 6 FACS Isolation of NP+ Splenic B Cells for Affymetrix and qPCR Analysis 4oC; NaAzide 0.05%; FCS Bonnie JhD-/- Jk-/- mice PI 35.1 50.8 # Cells 97.3 PE-anti-AA4.1 Side Scatter Naive Fitc-anti-B220 APC-NIP 97.9 51.7 APC-NIP 17.4 # Cells Side Scatter Memory 79.3 77.9 PI Fitc-anti-B220 Bonnie JhD-/- mice 12 weeks post 2nd immunization Biot-anti-Kappa Cell Surface Molecules: M>N (Affy and qPCR Confirmed Mela (80-kD Melanoma Antigen) Emp1 (epithelial membrane protein 1) Bmpr1a (bone morphogenetic protein receptor, type 1A) Atp11a (ATPase, class VI, type 11A) Myadm (myeloid-associated differentiation marker) Adora2a (adenosine A2a receptor) CD80 CD36 Acknowledgements • • • • • Tom, Geoff, Gouzel, and Don HHMI, Internal Medicine, YCCC YSM Administration Shannon Anderson and Mary Tomayko Today’s speakers