Attune NxT Acoustic Focusing Cytometer The next generation in acoustic cytometry

Attune NxT Acoustic Focusing Cytometer The next generation in acoustic cytometry Maybelline Giam Field Application Scientist The world leader in serving science

Attune NxT Flow Cytometer Attune NxT Acoustic Focusing Cytometer Acoustic-assisted hydrodynamic focusing to achieve freakishly fast flow rates 2

Introduction to Flow Cytometry Topics Module 1: Flow Cytometry Principles 3

What is Flow Cytometry? CYTOMETRY is the measurement of physical or chemical characteristics of cells or particles FLOW CYTOMETRY measurements are made as cells or particles in suspension pass individually through a flow cytometer instrument Performed on single cell suspensions Provides discrete measurements from each cell Provides a distribution of the measured characteristics 4

Advantages of flow cytometry Single cell data (great for heterogeneous populations) Measurements on large numbers of cells Rich statistical analysis on cell populations Ability to multiplex Thousands of cells can be analyzed rapidly Small sub-populations of cells can be identified In specific instruments, cells can be physically sorted. Sub-populations can be purified for further study 5

Some applications of flow cytometry Immunophenotyping (eg. B/T/NK cells in blood, immune organs) Immune assays (eg. T cell killing, phagocytosis) Cytokine production Stem cell characterisation (eg. CD34 HSCs) Analysis of transfected cells (eg. % GFP positivity) Phenotypic assays (eg. proliferation, apoptosis, ROS) Drug screening Protein expression (even phospho-proteins) RNA expression (eg. PrimeFlow) Small particle analysis (eg. bacterial, yeast) 6

The Flow Cytometer A Different kind of Microscope Data Analysis Laser Sample Flow cytometry is similar to a microscope. Microscope produces an image of a cell Light A flow cytometer collects and quantifies scattered light and fluorescence. The output produced is numbers. 7

8 Comparing flow cytometry and microscopy

Flow Workflow Sample prep Blood, tumor, cells Staining Flow Run 9

Parameters that Flow cytometry can measure Relative size Cell complexity/granularity Fluorescence Amount of DNA Levels of proteins (on cell surface or intracellular) 10

When the light hits the cells, two phenomena occur Before we talk about absorption and fluorescence emission, there is scattering of light! When laser light interacts with a cell, light is scattered in all directions. The light scatter depends on size and internal complexity of the cell. laser 11

Light scattering in human whole blood Large and granular Large, less granular Small, not granular 12

Light scattering differs when cells have different physiology Aged culture of Jurkat T cells: green are live cells & red are dead cells 13

What if 2 cell types are the same size and complexity? Side Scatter (SSC) Neutrophils Lymphocytes Monocytes Forward Scatter (FSC) 14

What if 2 cell types are the same size and complexity? CD19 CD3 B T Use cell markers to differentiate between cell types Markers can be: intracellular enzymes, surface receptors, adhesion molecules ANYTHING A FLUORESCENT DYE OR ANTIBODY CAN DETECT 15

Separate T and B lymphocytes by their cell surface markers B Cells Side Scatter (SSC) Neutrophils Lymphocytes Monocytes CD19 PE T Cells Forward Scatter (FSC) CD3 Alexa Fluor 488 16

What is spillover and compensation? BL1 BL2 17

What is spillover and compensation? BL1 BL2 What is the real R-PE fluorescence? Need to subtract A488 signal 18

What is spillover and compensation? Compensation is the process which corrects the detected "spillover" of the emission of one fluorochrome into the detector designed to collect the emission from another fluorochrome. The primary purpose is to allow the measurement of the true fluorescence in the fluorescence channel contaminated by the spillover. Use single color samples are used to set compensation 19

What is spillover and compensation? Un-Compensated Alexa 488 Single Color Control Alexa 488 is being detected by the PE channel as well! 20

What is spillover and compensation? 21

What is spillover and compensation? Properly Compensated Alexa 488 Single Color Control 22

Un-Compensated 1e6 Un-compensated FITC PE APC PerCP-Cy5.5 4_Intensity 1e5 1e4 0 400 450 500 550 600 650 700 Emission Wavelength (nm) Detector (nm) 530 575 660 700 1000 1000 1e4 1e5 3_Intensity FITC 1e6 Un-compensated PE 1e5 6_Intensity 1e4 APC 1000 0 1000 1e4 1e5 1e6 5_Intensity PerCP-Cy5.5 Data Courtesy Cancer Research UK 23

Compensated 1e6 Compensated FITC PE APC PerCP-Cy5.5 1e5 4_Intensity 1e4 1000 0-1000 -1000 0 1000 1e4 1e5 3_Intensity 0 400 450 500 550 600 650 700 Emission Wavelength (nm) Detector (nm) 530 575 660 700 FITC 1e6 Compensated PE 1e5 6_Intensity 1e4 1000 0-1000 -1000 0 1000 1e4 1e5 5_Intensity APC PerCP-Cy5.5 Data Courtesy Cancer Research UK 24

Auto-compensation in the Attune NxT 25

Auto-compensation in the Attune NxT 26

Principles of Panel Design 1. Understand fluorochromes and know your instrument. 2. Check for reagent availability. 3. Match fluorochromes by brightness 4. Minimize spillover 5. Use appropriate controls 27

Introduction to Flow Cytometry Topics Module 2: Flow Cytometer Hardware 28

What does a flow cytometer consist of? Flow Cytometry is a technology that simultaneously measures and analyses multiple characteristics of single particles as they flow in a fluid stream through a beam of light. 1. Fluidics - To introduce the cells for interrogation with laser 2. Optics - To generate and collect the light signals 3. Electronics - To convert the optical signals to electronic signals 29

What does a flow cytometer consist of? 30

Traditional flow cytometers use only hydrodynamic focusing Particle positioning in laser is important Low sample flow rate (e.g., 12 µl/min) Hydrodynamic core High sample flow rate (e.g., 200 µl/min) Focused laser Focused laser Narrow particle focus = Narrow distribution Broad particle focus = Broad distribution Intensity sheath sheath sheath sheath Count Count Intensity 31

Attune NxT uses Acoustic Assisted hydrodynamic focusing Hydrodynamic Sample Core High sample flow rate > 100 µl/min Acoustic Acoustic Focusing Assisted Hydrodynamic Followed Focusing by Hydrodynamic Focusing Focused laser Narrow Data distribution Sheath Count Intensity 32

Acoustic focusing End-on view of capillary 33

Comparable Results at All Flow Rates Traditional Cytometers Attune NxT µl/min µl/min 200 Up to10x Faster than Traditional Cytometers 12 µl/min 35 µl/min 60 µl/min 12.5 µl/min 25 µl/min 100 µl/min 200 µl/min 500 µl/min 1,000 µl/min CV= 4.83 CV=6.12 CV=7.76 CV= 2.99 CV= 3.03 CV= 2.76 CV= 2.94 CV= 2.70 CV= 2.96 Hydrodynamic Focusing Only Acoustically Enhanced Hydrodynamic Focusing 34

Acoustic Focusing Dilution is the solution 35

Rare Event Detection How many events are enough? For an acceptable level of significance, 5% CV, you need 400 positive cells In a sample with 1 x 10 6 cells/ml Frequency Total events Time for speed = 100ul/min Time for speed = 1000ul/min 10% 4,000 0.04 0.004 1% 40,000 0.4 0.04 0.1% 400,000 4 0.4 0.01% 4,000,000 40 4 0.001% 40,000,000 400 40 36

Some applications involving Rare Cell Analysis Counting of CD34+ hematopoietic progenitor cells Data courtesy of Prof. David Cousins, University of Leicester Dendritic cell analysis (eg. pdcs) 37

Sample preparation workflow for whole blood analysis Collect Sample Generic sample preparation workflow Total sample prep time ~1.5-2.5hrs Process to Single cell Suspension 5-10 min Lyse red blood cells or enrich for white blood cells 30-60min Stain 15-30 min Wash 5-10 min Wash 5-10 min Wash 5-10 min Add 1-2 hours for intracellular markers Acquire Samples No Lyse/No Wash sample preparation workflow Collect Sample dilute Lyse red X blood cells or enrich for Stain white blood 15-30 min cells 30-60min Wash X 5-10 min Wash X 5-10 min Acquire Samples 38

Intact whole blood detection of WBC Rational: Hemoglobin in RBCs absorbs light at 405nm while WBC and Platelets do not, allowing resolution using light scatter alone (no antibody staining needed) Strategy: Install Attune NxT No-Wash, No-Lyse Filter kit to allow dual-laser light scatter (SSC) detection using Blue and Violet laser 39

Results WBC detection in whole blood Normal FSC/SSC 40

Results WBC detection in whole blood SSC/VSSC Normal FSC/SSC 41

Results WBC detection in whole blood SSC/VSSC Normal FSC/SSC SSC (Violet 488 nm) (x10 6 ) FSC (Blue 488 nm) (x10 6 ) 42

Combining NWNL with unparalleled speed of Attune NxT No Lyse/No Wash assessment of phagocyte function in human blood 1. Incubate whole blood with phrodo BioParticles 2. Stain with Vybrant DyeCycle Ruby (threshold on nucleated WBCs) 3. Dilute and analyse on Attune NxT (flow rates 200µl/min) phrodo BioParticle Conjugates: ph-sensitive reagents that fluoresce upon their ingestion into acidic phagosomes 43

Combining NWNL with unparalleled speed of Attune NxT No Lyse/No Wash assessment of phagocyte function in human blood 44

On Board Fluidics Fluid storage: All fluids stored within instrument with active fluid level sensing Fluid Lines Sensor Connections Focusing Fluid Filters Standard fluidic tanks: 1.8 L focusing fluid tank 1.8 L waste tank 175 ml shutdown solution tank 175 ml wash solution tank Nominal fluid consumption: 1.8 L/day Waste Focusing Fluid Wash Fluid Shutdown Fluid Easy access to tanks for refilling 45

Smooth and Accurate Sample Delivery Sample delivery: Sample delivered by positive displacement syringe pump for volumetric analysis Volumetric cell counts: Live/dead analysis Sample analysis volume: 20 µl 4 ml Sample rates: 12.5 1,000 µl/min Unused sample return option: Sample returned into plate or tubes Syringe Pump Wide dynamic range Long life = 8 million cycles Easy syringe replacement 46

Attune NxT Cytometer is Clog Resistant Attune NxT uses a positive displacement syringe pump Samples are pushed through the system with a direct displacement (and volumetric) pump Pump pushes potential clogs through with pressure that builds steadily if there is a restriction Attune NxT flow cell is 200 um in diameter. This is relatively large in the industry Attune NxT flow path is back-flushed after every sample Back-flush is into an orifice that is larger than the sample path Any accumulation in the forward path is cleared back into a larger orifice after every sample. 47

What does a flow cytometer consist of? 48

Optical Configuration 49

Flat Top Lasers Maintain High Resolution When Stream Shifts Flat System top provides is misaligned stable and optical producing alignment large CV s Flow Cell 15 um beam shift 10 um Core Stream Measured CV > 1.7% 1.1% 10% Gaussian Laser Profile Plus: Lasers Only on During Acquisition! 10X Increase in Lifetime 50

51 Violet Blue Yellow Red - Standard Configuration 417LP 654LP 577LP Blank Blank Blank 710/50 780/60 780/60 Blank 503LP 695/40 603/48 Blank 695/40 512/25 720/30 620/15 440/50 670/14 585/16 Blank 590/40 530/30 488/10+OD2

695/40 574/26 530/30 488/10+OD2 52 Attune NxT Blue Violet Configuration Blue laser (488nm) 780/60 Blank 503LP 780/60 695/40 574/26 530/30 488/10

Utilizing four excitation sources limits spillover Having four excitation sources allows the user to select spectrally distinct fluorochromes, thus limiting spillover. CD62L Pacific Blue TM Violet 405 nm Blue 488 nm CD8 FITC CD4 PerCP Cy 5.5 CD56 PE Yellow 561 nm Red 638 nm CD3 APC-Alexa Fluor 750 53

Utilizing the full spectrum limits the need for compensation CD3 APC-Alexa Fluor 750 (RL-3) αβt cells NKT cells NK cells CD62L Pacific Blue TM (VL-1) CD62L Pacific Blue TM (VL-1) CD56 PE (YL-1) CD8 FITC (BL-1) CD4 PerCP Cy 5.5 (BL-3) Naïve and Central Memory Effector Memory Compensation Matrix Pacific Blue TM (VL-1) FITC (BL-1) PerCP Cy 5.5 (BL-3) PE (YL-1) APC Alexa Fluor 750 (RL-3) Pacific Blue TM (VL-1) 0.0 0.0 0.0 0.0 FITC (BL-1) 0.0 0.0 0.0 0.0 PerCP Cy 5.5 (BL-3) 0.0 0.0 0.0 0.0 PE (YL-1) 0.0 0.0 0.0 0.0 APC Alexa Fluor 750 (RL-3) 0.0 0.0 0.0 0.0 54

What does a flow cytometer consist of? 55

Electronics Convert optical signals (photons) to proportional electronic signals (voltage pulses) Electronic signals are processed by the on-board processor Analyze voltage pulse height, area, and width Interface with the computer for data transfer Schematic of a photomultiplier tube. The photon is converted to an electron when it enters the PMT at the cathode. It then travels through the PMT, being amplified at the dynodes (electrodes) throughout and end up at the anode which is a collecting electrode. 56

Electronics Sample Flow Voltage Pulse in PMT Laser Voltage Time Laser Voltage Time Laser Voltage Time 57

Adjust PMT Voltages Expand the Voltage section Adjust FSC & SSC voltages to position cell population on the scatter plot Adjust Fluorescence Channels voltages to position the auto fluorescence signal (unstained population) to ~10 3 Notes: If compensation controls are recorded, all the fluorescence channels voltages are disabled (i.e. grayed out) 58

Optimizing PMT voltage If PMT too low, might not be able to differentiate dim population from bright population If PMT too high, background will be very high and you might have spillover from other dyes PMTs need to be just right! Voltage walking 59

Sample Presentation: Voltage Pulse Pulse Area Volts Pulse Height 0 Pulse Width Time (µ Seconds) Attune default: All parameters (A-H-W) on all channels 60

Doublet discrimination In most cases, data analysis should include gating on single cells. Three Methods of Determination Pulse width double pulse height Pulse width double pulse area Linear Relationship of Pulse Height to Area 61

Thresholds Electronic threshold can be used to limit the number of events that the flow cytometer acquires Only signals with an intensity greater than or equal to the threshold will be processed and analysed X X 62

Introduction to Flow Cytometry Topics Module 3: Attune NxT Software 63

Main Application Workspace Ribbons Experiment Explorer Tabs Workspace 64

Experiment Explorer User name Experiment name Compensation controls Group name Sample name (highlighted in orange = active) 65

Tabs Instrument Settings Tab Select and label channels Change PMT voltage 66

Two Ribbons of Note Workspace Ribbon Plot Options Gating Tools Statistics box Format Layout Instrument Ribbon Fluidic functions Access PT results 67

Histograms A histogram tells us the number of events (or cells) at a particular measurement value 68

Dual Parameter Plots Dot Plots Back-gating 69

Dual Parameter Plots Density Plots Precedence density plots (Density + Back-gating) 70

Sequential gating to identify populations Gate Sample name Y axis X axis Statistics Box 71

Easy data comparison using overlays Selected plots within a single experiment are superimposed to allow direct visual comparison of data Single parameter: Histograms Percent of Max Dual parameter: Dot/Density Plot 72

Easy data comparison using overlays Selected plots within a single experiment are superimposed to allow direct visual comparison of data Single parameter: Histograms Percent of Max Dual parameter: Dot/Density Plot 73

Analysis using heatmaps 74

Shutdown with one click 75

Attune NxT Acoustic Focusing Cytometer Modular Design in a small footprint 4 lasers with 16 detection channels FAST Unprecedented Acoustic Focusing Technology Highest speed is ~10X faster than max speed of traditional flow cytometers DOES NOT CLOG Robust performance Reduced Clogging and Flat top lasers (less downtime) Simplifies sample prep CELL COUNTING Volumetric, no beads Sample Recovery High-throughput Plate Autosampler Compatible with standard and deep well 96/384-well plates 76

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