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The BD Accuri C6 Flow CytometerA powerful system with a simple design.Charlie Hsu 許博彥Instrument Product Specialist騰達行/Unimed Healthcare INC.
BD Biosciences Cell AnalysisAccuri’s MissionProliferate thepower of flow cytometryto every lab that works withcells, beads, or particlesworldwide.
BD Biosciences Cell AnalysisAccuri is Enabling Discovery3
BD Biosciences Cell AnalysisThe C6 Flow CytometerInnovations in all the major components of a flow cytometer Fluidics Optics Electronics Software
BD Biosciences Cell AnalysisPrinciple of Flow CytometryComputerFluidicsOpticsElectronics
BD Biosciences Cell AnalysisAccuri Innovation - Fluidics Two peristaltic pumps operating: push-pull system– Sheath Pump: pushs fluid towardsflow cell– Waste pump: pulls sheath andsample past flow cellLaminar flow :hydrodynamic focusingPatented pulse dampenersVolume measurement for absolute countsUp to 10,000 events/second
BD Biosciences Cell AnalysisCell Count Verification for the Accuri C6Coefficient of VariationCounting Bead: cell count per mL4010,000,000HemacytometerHemacytometerp 0.002y 1.116xr² 0.9774n meter)1010,000counting beadsCountingbeads(C6 cytometer)p 0.0101,0001,0001210,000100,00031,000,000Cell Counting Method10,000,000Direct Volume: cell count per mL sampleThe average coefficient of variation for replicate cell counts using three different countingmethods on the same samples.A paired student’s T test was used to determine p values (95% confidence, N 23).7
BD Biosciences Cell AnalysisC6 Maximum Event Rate 10,000 per sec
BD Biosciences Cell AnalysisUnique Fluidics System Simplifies SampleHandling Microprocessor-controlled peristaltic pumps enabledirect volume measurement Many types of sample tubes may be used No need to transfer samples Save time and materials Add reagents or cells during a run With CSampler: culture, stain and run, all in oneplate9
BD Biosciences Cell AnalysisAutomated Flow Cytometry. Now It’s Easy.CSampler Add-on automation for the C6 10Flow CytometerProcesses 96-well and 48-wellplates24-position rack for 12 x 75 mmtubesPriority interrupt for urgentsamplesEasy-to-use software
BD Biosciences Cell AnalysisAccuri Innovation –Pre-optimized and locked-down optics Cylindrical flow cell 488 nm solid state laser640 nm diode laser Allows for fluorescence detectors at any location– No dichroic mirrors utilizedbetween flow cell and detectors– Increased signal strength to PMT– Reduces alignment issues Results in a very stable system that is easy to transport withoutalignment issues
BD Biosciences Cell AnalysisAdvantages of Pre-optimzed Detector Settings No specialist training or dedicated operator required Predictable, reproducible analysis relative to sample type and application Saves time and sample Attenuation filters (for too-bright signals) give controlled signal reduction Predictable fluorescence spill-over Focus on the science of measuring fluorescence, not the art of settingvoltages12
BD Biosciences Cell AnalysisAccuri Innovation –Electronics with Broad Dynamic Range18-Bit SystemZoom AreaZoom Area24-bit Accuri C6Beads: 1, 2, 12, 29 micronMore than 6 decades of signal on a single scaleAll your data, available at any time13
BD Biosciences Cell AnalysisPre-Optimized Light Scatter DetectorsAdjust Your View with ZoomZoom AreaLymphocytesZoom AgainZoom Out
BD Biosciences Cell AnalysisAccuri Innovation – Intuitive Software15
BD Biosciences Cell ustry Immunophenotyping Cellular processes: viability, apoptosis,membrane potential changes, ROS, ion fluxes Cell counting Protein expression and activation DNA content/ploidy/cell cycling Cytokine detection – intracellular and excreted Multiplexed protein quantitation (BD CBA beads) Disease diagnosis/ treatment monitoring Cord and blood product quality Bacteria, yeast detection, viability, quantitation Algae/ phytoplankton analysis16
BD Biosciences Cell AnalysisHuman PBMC : T cell CIsotype-FITCCD45RA-FITCGate:CD3 CD4 CD4-PELymphocyte GateGate:CD3 CD8 CD3-APC17
BD Biosciences Cell AnalysisAbsolute counts of viable CD8 and CD4 T cells:Mouse splenocytesGate P1Volume ( μL) 0886.46.46.46.49154Total Cells in P15,9036.4922Viable CD8 CellsCD8a PE-ACD8a PE-AGate P17-AADLight Scatter GateGated on (P1 in all)This PlotQ8-ULQ8-URQ8-LLViable T Cells: Lower Right QuadrantCountGated on (P1 in all)This PlotQ9-ULQ9-URQ9-LLQ9-LRCD4 FITC-AVolume ( μL) 9154Total Cells in P17,8756.41230Viable CD4 Cells
BD Biosciences Cell AnalysisProliferation Assay--CFSEDivisions:3210Divisions:3 2 10Lyons and Parish, 1994JIM, 171;131CFSEFL-1 (Log)
BD Biosciences Cell AnalysisIn Vitro T cell proliferation:T cells dendritic cells (DC)Gate CD3 Cells% of CD3 T cellsProliferating0.4%DC T cells1:4FSC-AT cells only52.4%CFSECourtesy of: Reddy P, Sung Y. Department of Pediatrics, University of Michigan, Ann Arbor, MI20
BD Biosciences Cell AnalysisUse of 7-AAD for Viability DeterminationViable cells within higherFSC region areimpermeable to 7-AADDead cells within lowerFSC region arepermeable to 7-AADMethods: Splenocytes from 3 to 6-month old C57BL/6 mice wereirradiated with UV light. Cell viability was determined by adding 1 µL 7AAD from a stock solution of 1 mg/mL (BD PharmingenTM) per 100 µLvolume of cells.7-AAD
BD Biosciences Cell AnalysisDNA Detection and Cell Cycle AnalysisData File Format: FCS 3.0 compliantCompatible with: FCS Express: CFlow FileImporter MultiCycle FlowJo 7.6 WinlistTM and Modfit LTTM VenturiOne 22
BD Biosciences Cell AnalysisDNA Staining with Propidium IodideControl (DMSO)G1HU-331Sub - G1SG2/MMethods: Jurkat cells were treated with either DMSO (Control; 2%) or HU-331, anapoptosis inducing cannabinoid (5 mg/mL), Cayman Chemical Company, for 6 hours. Cellswere then fixed and stained with Accuri Cell Cycle Phase Determination Kit (KR-300).
BD Biosciences Cell AnalysisCalcium Flux Measurement with Fluo-4Sample tubes on the C6 do not require pressurization. Agonists can be added during sample acquisition,ensuring that one is able to visualize the entire kinetic activity.
BD Biosciences Cell AnalysisFluorescent Protein Detection with the Accuri C6Transfection ScreeningGFP25
BD Biosciences Cell Analysisgp64-PE Expression in SF9 Insect CellsInfected with Baculovirus.gp64-PEVirus GenerationP2P3Gp64-PEgp64-PEFSC-HViralDilutionNo virus1:101:100261:1000
BD Biosciences Cell AnalysisThe Accuri C6 is Robust!The Ecosystem Centre:Palmer Peninsula, AntarcticaNational Oceanic and Atmospheric Administration:Great Lakes Microcystis research project – Lake Erie2nd Norwich Flow Day:Back of Kate’s car, Institute for Food Research27
BD Biosciences Cell AnalysisThanks for Your Attention許博彥 Charlie HsuInstrument Product SpecialistMail: poyenhsu@unimed.com.twTEL: 02-27202215 Ext:241Web: www.unimed.com.tw
Cell Count Verification for the Accuri C6 y 1.116x r² 0.9774 n 75 1,000 10,000 100,000 1,000,000 10,000,000 1,000 10,000 100,000 1,000,000 10,000,000 mL Direct Volume: cell count per mL sample Cell Counting Method 1 2 3 ation 0 10 20 30 40 direct-volume (C6 cytometer) counting beads (C6 cytometer) Hemacytometer Accuri C6 Counting beads p .
