Cytek Biosciences Aurora User manual

Aurora™ User’s Guide

Copyrights
©2017, Cytek Biosciences Inc. All rights reserved. No part of this publication may be reproduced, transmitted, tran-
scribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means:
electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from Cytek
Biosciences.
The information in this guide is subject to change without notice. Cytek Biosciences reserves the right to change its prod-
ucts and services at any time to incorporate the latest technological developments. Although this guide has been pre-
pared with every precaution to ensure accuracy, Cytek Biosciences assumes no liability for any errors or omissions, nor
for any damages resulting from the application or use of this information. Cytek Biosciences welcomes customer input
on corrections and suggestions for improvement.
Trademarks
Cytek, the Cytek logo, and all other trademarks are property of Cytek Biosciences. © 2017 Cytek
FCC Information
WARNING: Changes or modifications to this unit not expressly approved by the party responsible for compliance could
void the user's authority to operate the equipment.
NOTICE: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part
15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the
equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency
energy and, if not installed and used in accordance with the instruction manual, can cause harmful interference to radio
communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case
the user will be required to correct the interference at his or her own expense.
Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits.
This Class A digital apparatus meets all requirements of the Canadian Interference-Causing Equipment Regulations.
Cet appareil numérique de la classe A respecte toutes les exigences du Réglement sur le matériel brouilleur du Canada.
CDRH Information
This equipment complies with CDRH Class I requirements.
Regulatory Information
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
History
Revision Date Change
52-70001-0A 10/2017 Initial release

iii
Chapter 1: Introduction 7
About this Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Safety Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
General Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Biological Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Chapter 2: Overview 9
Cytometer Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Software Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Spectral Unmixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Understanding Full Spectrum Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Reference Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Chapter 3: Startup & Shutdown 17
Filling the Sheath and Emptying the Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Filling the Sheath . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Emptying the Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Starting Up the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Shutting Down the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Chapter 4: QC & Setup 21
Daily QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Performing Daily QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
QC Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Instrument Setup - Reference Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Creating Reference Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Running Reference Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Updating Reference Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Levey-Jennings Tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Gain Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Alarm Ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Chapter 5: Acquisition 31
Raw vs Unmixed data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Unmixing and Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Contents

iv Aurora User’s Guide
Setting Up an Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Experiment Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Creating a New Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Unmixing Workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Live Unmixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Post-Acquisition Unmixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Chapter 6: Advanced Unmixing 47
Unmixing in the Analysis Workspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Virtual Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Chapter 7: Library, Preferences, and Users 55
Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Fluorescent Tags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
User Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Worksheet Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Experiment Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Worksheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Fonts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Notifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
QC Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Managing Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Use Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Chapter 8: Maintenance 71
Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Scheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Unscheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Cleaning the SIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Purging the Sheath Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Removing Air Bubbles from the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Cleaning the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Decontaminating the Fluidics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Cleaning the External Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Inspecting the Fluidics Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Replacing the Sheath Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Replacing the SIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

v
Chapter 9: Troubleshooting 81
Chapter 10: Glossary 83
Chapter 11: Specifications 87
Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Fluorescence Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Installation Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Chapter 12: Supplies and Replacement Parts 91

vi Aurora User’s Guide

Chapter 1: Introduction 7
1
Introduction
About this Guide
This manual provides information on the Aurora flow cytometer, daily workflow, SpectroFlo™
software features, cytometer specifications, and instrument maintenance. It also includes
troubleshooting tips and service information.
Safety
Safety Symbols
The Aurora is intended for research use only. Not for diagnostic or therapeutic procedures.
The following table lists symbols used throughout this guide.
General Safety
• Do not place any object on top of the instrument.
• Before turning on the cytometer, visually inspect all containers. Wear the recommended
protective laboratory attire such as protective gloves, eyewear, and lab coat.
• Purge the sheath filter if air bubbles are visible in the sheath filter, or if the plenum or sheath
container have run dry.
• Fill the sheath container as needed. Never use tap water as sheath solution.
• Do not run bleach or detergent through the sheath filter. It is difficult to remove cleaning
solutions from the sheath filter.
Symbol Meaning
Caution: hazard or unsafe practice that could result in material damage,
data loss, minor or severe injury, or death
Risk of electric shock
Biological risk

8Aurora User’s Guide
• Check the cytometer periodically for fluid leaks or crimped lines. If evidence of a leak is
detected, contact Cytek Technical Support immediately. Do not attempt to repair the
instrument.
• When performing daily QC, always select the correct bead lot number.
Electrical Safety
• Do not place liquids on top of the instrument. Any spill into the ventilation openings could
cause electrical shock or damage to the instrument.
Biological Safety
• Empty the waste container when filling the sheath container or as needed to prevent leakage.
Take care to avoid damaging the fluid level sensor in the waste tank.
• Biological samples are potentially dangerous and/or life threatening. Adhere to proper
handling procedures for samples and reagents. Wear appropriate laboratory attire such as
protective gloves, eyewear, and lab coat.
• Any instrument surface in contact with biological specimens can transmit potentially fatal
disease. Use universal precautions when cleaning the instrument or replacing parts.
• Concentrations of sodium hypochlorite higher than 10%, as well as other cleaning agents can
damage the instrument.
Technical Support
For instrument support within the US, call 1-877-92-CYTEK. Visit our website, www.cytekbio.com,
for up-to-date contact information.
When contacting Cytek, have the following information available:
• Serial number
• Any error messages
• Details of recent performance

Chapter 2: Overview 9
2
Overview
Aurora System
The Aurora system consists of the Aurora flow cytometer and a computer workstation running
SpectroFlo™ software for acquisition and analysis. The system also includes SpectroFlo QC beads.
The cytometer is an air-cooled, compact benchtop instrument. It is equipped with three lasers and
up to 48 detection channels for fluorescence, and up to two detection channels for scatter (FSC and
violet laser SSC). Sheath and waste fluids are contained in either 4-L tanks or 20-L cubitainers.
During operation the software notifies you when the fluid levels are getting high or low. The
pressurized fluidics system includes a plenum for storing sheath, allowing you to fill and empty the
fluids during operation.
The workstation is a dedicated USB-compatible PC with monitor, keyboard, and mouse. It runs
Microsoft® Windows® 10 Pro 64-bit operating system, which is required for SpectroFlo software.
Figure 1. Aurora System

10 Aurora User’s Guide
Cytometer Overview
The Aurora spectral flow cytometer is an air-cooled, multi-laser, compact benchtop flow cytometer.
It is equipped with three lasers and up to 48 detection channels for fluorescence and up to two
detection channels for scatter (FSC [forward scatter] and violet laser SSC [side scatter]). Solid-state
lasers transmit light through a flow cell where particles in suspension are focused, single file for
interrogation by the laser. Proprietary, high-sensitivity, 16-channel semiconductor detector arrays
are equipped to capture the emission spectra of dyes that emit in the 400 to 900-nm wavelength
range. The resulting fluorescence and scatter are then collected and converted into electronic
signals. On-board electronics convert these signals into digital data that can be acquired and
recorded on the workstation.
The cytometer power button is located on the left side of the upper panel of the cytometer
(Figure 2). When the cytometer is powered on, the power button is illuminated.
The front panel opens on its hinges to the right to reveal the fluidics system. The top cover opens to
reveal the optics.
Front of Cytometer
Figure 2. Aurora front and left side panels
Do not place any object on top of the instrument.
Do not place liquids on top of the instrument. Fluid leaking into the cytometer could
cause electrical shock or damage to the instrument.
SIT door

Chapter 2: Overview 11
Back of Cytometer
Allow 12.7 cm (5 in) between the back of the cytometer and the wall for proper ventilation.
Figure 3. Back of cytometer
Fluidics
Sample Injection Port/Sample Injection Tube
Sample, contained in a standard 12 x 75-mm tube, enters the cytometer through the sample
injection tube (SIT) that is contained within the sample injection port (SIP) [Figure 4]. The sample
tube snaps into place under the SIP requiring no additional tube retention support. The SIT extends
from the SIP during acquisition and retracts when the cytometer is not acquiring.
Figure 4. Sample injection port and sample injection tube
Fluid Containers
The Aurora draws sheath solution directly from a 20-L sheath cubitainer or the 4-L sheath tank
provided by Cytek. It expels waste into an empty 20-L cubitainer or the 4-L waste tank provided by
Cytek.
USB connection to workstation
power cable
mains power switch
SIT
SIP

12 Aurora User’s Guide
The fluidics tanks are contained in a holding reservoir located on the left side of the cytometer
(Figure 5). The 4-L tank with the transparent fluidic line is for sheath solution. The 4-L tank with the
orange fluidic line is for waste.
Figure 5. Aurora fluidics bottles and front panel
Fluid Flow
The Aurora fluidics is driven by vacuum. An accumulator vessel is the source of vacuum for the
system. Sheath solution is drawn into and stored in the sheath plenum before passing through a
sheath filter where debris and contaminants are removed. Before reaching the flow cell, the sheath
stream passes through a degasser, which removes air bubbles. After passing the laser
interrogation point, the combination of sheath solution and sample travels to the waste container.
Sheath and waste fluid levels are monitored by sensors. The waste level sensor is located
underneath the waste tank cap. The sheath level sensor is located underneath the sheath plenum
cap. Both sensors are monitored by the software.
Figure 6. Aurora fluidics overview

Chapter 2: Overview 13
Fluidics Components
The following figure shows the fluidics components.
Figure 7. Fluidics components (inside fluidics compartment)
The following table describes the fluidics components.
No. Component Description
1 Plenum pump Pulls sheath from the sheath tank to fill the plenum
2 Vacuum pump Maintains the vacuum in the accumulator
3 Plenum Storage vessel for sheath fluid before it flows to the sheath filter
4 Degasser Removes air bubbles from the sheath fluid
5Sheathfilterquick
connects (x3)
Sheath filter fluid input, fluid output, and vent line quick-connects
6 Sheath filter Filters debris and particles from the sheath fluid
7 Accumulator Vacuum source for the fluidics system
7
6
5
4
3
2
1

14 Aurora User’s Guide
Optics
Unlike conventional flow cytometers that direct specific bandwidths of fluorescence light into
discrete detectors or photomultiplier tubes (PMTs), the Aurora uses a solid-state, multi-channel,
narrow-beam detector array for each laser. Each array can be configured with up to 16 detectors
that are used to capture a part of the emission spectrum from each particle passing through the
laser beam. The detector channels from all three lasers are used to capture the entire emission
spectra from each fluorescent-labeled particle. Spectral deconvolution (unmixing) algorithms
calculate the contribution of the known individual fluorophore’s spectra to the total collected
signal.
Figure 8. Optical schematic
The default optical configuration has 16 channels for detection off the violet laser, 14 channels off
the blue laser, and 8 channels off the red laser. Detectors are referred to as V1–V16, B1–B14, and
R1–R8, for the violet, blue, and red lasers, respectively. The wavelengths detected by each detector
(channel) increase across the array. See the table on page 52 for details.
For excitation, a proprietary flat-top laser design enables a constant power distribution across the
width of the flow cell.

Chapter 2: Overview 15
Software Overview
SpectroFlo software allows you to acquire and analyze samples and adjust instrument settings.
Once you log into the software, a Get started menu appears with six modules from which to
choose.
Six options provide workspaces that allow you to perform various functions.
Module Description
QC & Setup Daily QC ensures that the instrument is in optimal condition for use. Run
SpectroFlo QC beads daily to assess system performance and allow the software to
adjust settings for day-to-day variation. Levey-Jennings reports keep track of trends
in system performance. Setup allows you to create Reference Controls. See “QC &
Setup” on page 21 for more information.
Acquisition The Acquisition workspace allows you to create experiments to acquire and
analyze data. Experiments can be created through a guided wizard or created from
previously saved templates. See “Acquisition” on page 31 for more information.
Analysis Here, FCS files can either be unmixed or compensated using virtual filters. See
“Advanced Unmixing” on page 47 for more information.
Library The Library allows you to store experiment templates, worksheet templates, user
settings, fluorescent tags, SpectroFlo QC bead information, and label information.
See “Library” on page 55 for more information.
Preferences Software preferences can be changed to customize the software. Default plot sizes,
fonts, gate colors, print layouts, statistics box table option, and more can all be
changed in the Preferences. See “Preferences” on page 59 for more information.
Users The Users workspace contains user management options and administrative
controls. See “Users” on page 67 for more information.

16 Aurora User’s Guide
Spectral Unmixing
Spectral unmixing is an important concept to understand how data is generated and analyzed
using the Aurora flow cytometer with SpectroFlo software. Spectral unmixing is used to identify the
fluorescence signal for each fluorophore used in a given experiment.
Understanding Full Spectrum Flow Cytometry
Because fluorophores emit light over a range of wavelengths, optical filters are typically used to
limit the range of frequencies measured by a given detector. However, when two or more
fluorophores are used, the overlap in wavelength ranges often makes it impossible for optical
filters to isolate light from a given fluorophore. As a result, light emitted from one fluorophore
appears in a non-primary detector (a detector intended for another fluorophore). This is referred to
as spillover. In conventional flow cytometry spillover can be corrected by using a mathematical
calculation called compensation. Single-stained controls must be acquired to calculate the amount
of spillover into each of the non-primary detectors.
The Aurora's ability to measure a fluorochrome’s full emission spectra allows the system to use a
different method for isolating the desired signal from the unwanted signal. The key to differentiate
the various fluorochromes is for those to have distinct patterns or signatures across the full
spectrum. Because the system is looking at the full range of emission of a given fluorochrome, and
not only the peak emission, two dyes with similar emission but different spectral signatures can be
distinguished from each other. The mathematical method to differentiate the signals from multiple
fluorochromes is call spectral unmixing. Just as for compensation, single-stained controls,
identified in SpectroFlo software as Reference Controls, are still necessary, as they provide the full
fluorescence spectra information needed to perform spectral unmixing.
Spectrum plots from conventional spectrum viewer shows heavy overlap between Qdot 705 and BV711.
Spectrum plots from Aurora show distinct signatures for Qdot 705 and BV711.
Reference Spectra
Reference Controls, obtained by running single-stained and unstained samples, provide the
individual fluorescence spectra necessary to unmix the data. Either beads or cells can be stained
for use as Reference Controls. These controls can be acquired in the Reference Group of the
experiment during acquisition, or they can be acquired as Reference Controls in the QC & Setup
workspace. If Reference Controls are acquired in the QC & Setup workspace, they are stored and
can be used as Reference Controls for subsequent experiments.

Chapter 3: Startup & Shutdown 17
3
Startup & Shutdown
Filling the Sheath and Emptying the Waste
The color-coded sheath and waste quick-connects and the waste level sensor connector are
located at the lower-left corner of the front panel.
Figure 9. Sheath and waste line quick-connect and waste level sensor
Filling the Sheath
Fill the sheath container with manufacturer-provided sheath solution, MilliQ™ water, phosphate-
buffered saline (PBS), or DI water.
Sheath can be drawn from either the supplied 4-L sheath tank or directly from a 20-L cubitainer.
Sheath solution can be added to the sheath container while the instrument is running. The plenum
provides 5 minutes of run time at any flow rate while the tank is being filled or replaced.
Before turning on the cytometer, visually inspect all containers. Wear the recommended
protective laboratory attire such as protective gloves, eyewear, and lab coat.
Fill the sheath container as needed. Use only the appropriate sheath solution. Never
use tap water in the sheath container.
sheath line quick-connect
waste level sensor
waste line quick-connect

18 Aurora User’s Guide
Filling Sheath into a Cytek 4-L Sheath Tank or a 20-L Cubitainer:
1Remove the sheath fluidic line cap from the cubitainer or sheath tank lid from the Cytek sheath
tank.
2Add the appropriate sheath solution.
3Replace the fluidic line cap or sheath tank lid. Do not over-tighten.
4If the cytometer is powered on and the software is connected, verify that the software sheath
indicator is green.
Emptying the Waste
Waste can be expelled into either the supplied 4-L waste tank or directly into an empty 20-L
cubitainer.
Removing Waste from a Cytek 4-L Waste Tank or a 20-L Cubitainer:
1Disconnect the waste line quick-connect from the front of the cytometer.
2Disconnect the waste line orange quick-connect from the cubitainer cap or 4-L waste bottle.
Disconnect the waste level sensor.
The waste level sensor connector for the cubitainer is on the cubitainer cap. The waste level
sensor connector for the 4-L tank is on the front of the cytometer.
3Remove the waste cap from the cubitainer or the lid from the 4-L waste tank, taking care not to
damage the liquid level sensor.
4Dispose of the waste per local regulations.
5Add 2 L of undiluted bleach to the waste cubitainer, or 400 mL of bleach to the waste tank.
6Replace the waste cap/lid to the container. Hand-tighten the cap/lid until it is fully closed.
7Reattach the waste line and level sensor line to the cap/lid and front of the cytometer.
8If the cytometer is powered on and the software is connected, verify that the software waste
indicator is green.
Empty the waste container when filling the sheath container or as needed to prevent
leakage. The software indicator for waste will be yellow or red when the container needs
to be emptied. Take care to avoid damaging the fluid level sensor in the waste tank.
Biological samples are potentially dangerous and/or life threatening. Adhere to proper
handling procedures for samples and reagents. Wear appropriate laboratory attire such
as protective gloves, eyewear, and lab coat during this procedure.
Always treat the contents of the waste container with bleach (10% of the total volume).
Contents of the waste container may contain biohazardous material.

Chapter 3: Startup & Shutdown 19
Starting Up the System
1Turn on the workstation, then turn on cytometer.
NOTE: Ensure that a tube of DI water is loaded on the SIP before launching SpectroFlo
software. The tube is required for the SIT depth calibration.
2Launch SpectroFlo software and log in.
The cytometer initialization procedure begins. Sheath fluid is flushed through the fluidics lines
to eliminate any saline buildup. The system calibrates the SIT depth and the sample flow rate.
3Select QC & Setup from the Get Started screen.
4Check the status indicator in the lower-right corner of the screen. Ensure the indicator for
Connected is a green checkmark. If the indicator shows the instrument is not connected, check
to ensure that the USB connection between the cytometer and workstation is plugged into the
appropriate ports. See “Back of Cytometer” on page 11.

20 Aurora User’s Guide
5Check the sheath and waste level checkmark indicators. Ensure the status checkmark indicators
for sheath and waste are green before proceeding.
6The cytometer is now ready for Daily QC. See “Performing Daily QC” on page 21.
Shutting Down the System
The shutdown procedure flushes the flow cell and sample lines with bleach and DI water. The
software provides instructions during the shutdown procedure. The SIT will remain protracted
from the SIP at the end of the shutdown procedure to ensure the SIT does not dry and form clogs.
1In the Cytometer menu from either the QC & Setup or Acquisition workspace, select Fluidics
Shutdown.
2Place a tube containing 3 mL of 10% bleach on the SIP. Once loaded, the instrument begins
drawing in the tube contents. This takes approximately 2 minutes.
3When prompted, remove the tube and place a tube containing 3 mL of DI water on the SIP.
Once loaded, the instrument begins drawing in the tube contents. This takes approximately
2minutes.
4Leave the tube of DI water on the SIP. Make sure the SIT is submerged in the DI water at the end
of the Fluidics Shutdown procedure.
5Exit SpectroFlo software by clicking the X in the upper-right corner of the application window.
6Turn off the cytometer and workstation.
Fluid Indicator Meaning
Yellow sheath indicator Sheath tank is low and requires refilling (see “Filling the Sheath” on
page 17).
Red sheath indicator Sheath tank is empty and requires refilling (see “Filling the Sheath”
on page 17).
Yellow waste indicator Waste tank is nearing capacity and requires emptying (see
“Emptying the Waste” on page 18).
Red waste indicator Waste tank is full and requires emptying (see “Emptying the
Waste” on page 18).