Stem Cell Instrumentation Foundry
SCIF Orientation Flow Cytometry
Venu Polineni
Research Associate,
Stem Cell Instrumentation Foundry
UCM 5200 N. Lake Road Merced, CA 95343
Email: vpolineni@ucmerced.edu
Stem Cell Instrumentation Foundry
Overview of Flow Cytometry
Part I: Cell Preparation
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Get Single Call Suspension
Choose Fluorochromes
Label Cells with Fluorescent Probes
Write A Protocol
Set Up the Experiment
Part II: Instrumentation
• Sample Station: Sheath Pressure, Sample Pressure
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Flow Chamber: Sheath Fluid, Hydrodynamic Force
Lasers: 488nm, 633nm, 405nm, 315-355nm, 531-561nm (mW)
Detectors: Pick-up Lens, Optical Filters, PMTs
Flow Data: Flow Software, Listmode Files
Sorting: Drop Drive Frequency, Amplify, Phase charge, Delay
http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html
Stem Cell Instrumentation Foundry
Flow Cytometry Applications
• Cell Surface Analysis
-Fluorescence Surface Marker Labels
-Cell Toxicity-Lipid Peroxidation
• Intracellular Analysis
-Fluorescence Proteins: Gene Reporter; Enzymatic Assays;
-Cell Cycle: Hoechst 33342 (Live); PI; BUDR; AO
-Cell Signaling: Phospho-Specific Antibodies
-Cell Division by Dye Dilution: CFSE
-Hoechst 33342 to Identify Hematopoietic Stem Cells (SP)
• Functional Assays
-FRET (Fluorescence Resonance Energy Transfer)
-Intracellular Ca/Ph; Cell Viability
-Drug Discovery & Development: Apoptosis (BAX, FAS, BCL2, Fas…)
-Oxidative Metabolism: Using DCFH-DA Detect H2O2
-Measuring Mitochondrial Membrane Potential with JC-1 probe
• Sorting
-Transplantation
-Single Cell Cloning
-Sort Mitochondria, Chromosomes, Nuclei, Particles
-Purify a Specific Cell Population for Further Investigation
-Stem Cell Researches
• Microbiology
-Ocean Bacteria Study, Yeast, Virus
Stem Cell Instrumentation Foundry
SCIF Flow Cytometry Instrumentation
BD LSRII Multi-Laser Analyzer
BD AriaII/III Multi-Laser Sorter
Stem Cell Instrumentation Foundry
SCIF Flow Cytometry Instrumentation
LSRII: Analyzer
Aria II: Cell Sorter
Stem Cell Instrumentation Foundry
SCIF Flow Cytometry Instrumentation
Aria III Cell Sorter
Stem Cell Instrumentation Foundry
Safety
•-General Lab Safety
•-Electrical Safety
•-Optical/Laser Safety
•-Biohazard Safety
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Symbols
Electrical Shock
Caution
Laser
Irradiation
Biohazard
• Electrical Shock-Risk of Electric Shock
• Laser Irradiation-Avoid looking directly into
laser, as it may cause permanent eye damage.
• Biohazard-Biological hazard/Risk
• Caution-Important; Attention; Refer to
Accompanying Documentation
Stem Cell Instrumentation Foundry
General Lab Safety
The Flow Core is a BSL-2 Lab
• Controlled access
• Users must wash their hands after working with
potentially hazardous materials and before leaving the
laboratory.
• No food and drink
• Mouth pipetting is prohibited
• Perform all procedures to minimize the creation of
splashes and/or aerosols.
• Gloves, lab coat and eye protection be worn when
handling biohazard samples and waste fluid which may
contain hazardous levels of biological contamination
• Decontaminate Machine after completion of work
• Decontaminate all cultures, stocks, and other potentially
infectious materials
Stem Cell Instrumentation Foundry
Electrical Safety
• Stream Charge
When the sheath stream is charged and individual droplets
are formed, the droplets remain the charge present on the
stream.
 Do not insert any object into the charged stream.
 Do not touch any steel parts.
• Drop Drive Voltage
This ranges from 0-140 VAC and is used to drive the
piezoelectric crystal mounted in the cuvette flow cell
 Do not touch the cuvette flow cell
• Sort Deflection Plates
The range of voltage applied to these plates is 0-4000 VDC
 Do not touch the charged plates when HV is on.
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Electrical Charge of Sorting
Stem Cell Instrumentation Foundry
Laser Safety
• Accessible Emission Limit, AEL, Maximum Permissible Exposure,
MPE, Nominal Hazard Zone, NHZ, Laser Product Hazard
Classified, Class 1 to 4.
• Class1
• Meaning: safe
• Type of laser: very low power lasers or enclosed lasers.
• MPE: is never exceeded, even for very long exposure (hours), or
with the use of optical instruments.
• Nominal Hazard Zone: none.
• Typical AEM: 40 µW for blue.
• The lasers on LSRII and Arias are a class 1 product; meaning
operators are not exposed to harmful levels of laser irradiation
during normal operation.
• Cautions:
• Remove all jewelry when working with an open beam. Do not place
shiny or reflective objects into the path of the laser beam. Use all
protective housing , interlocks, and shields as identified in training.
Stem Cell Instrumentation Foundry
Biological Effects of Laser
Irradiation
• Eye Injury
eye exposure to a direct laser beam can cause
permanent eye damage including blindness. Laser
wavelengths between 400-1400 nm are the most
hazardous for retinal eye injury. UV-A lasers (315390 nm) can cause damage to the lens of the eye
contributing to cataracts.
• Do not expose your eye to the
horizontal plane of the laser beam
(direct or diffuse).
Stem Cell Instrumentation Foundry
Biological Effects of Laser
Irradiation
Skin Injury
• Skin exposure to direct and diffuse laser light can
cause damage. Lasers in the UV-A (315-390 nm) can
cause erythema (sunburn). Exposure in the UV-B
(280-315 nm) can cause the most severe effects,
such as sunburn, skin cancer and accelerated skin
aging.
• Wear protective clothing (lab coat,
long-sleeves) when using UV lasers
and when potential exposure to
direct laser beam exit.
Stem Cell Instrumentation Foundry
Biohazard Safety
IMPORTANT
If any hazardous organism, material, or agent is
used in the instrument, the Principal
Investigator is responsible for informing the
Dept. SCIF in writing of those hazards before
receiving service. This includes a list of all
pathogenic cell lines, hazardous reagents,
radioactive material, or agents with a BSL level
II or higher. Failure to report this information
may delay services on an instruments.
Stem Cell Instrumentation Foundry
Handling Biohazard Samples
Flow Core is a BSLII
• No Human Primary Cells Unless
-Tested NEGATIVE for all known infectious agents
-Fixed
• No Radioactive Samples
• Biohazard Samples
▫ Human Samples/Cell Lines
▫ Transgenic Mouse Samples
▫ Recombination DNA Samples
• Empty sample tubes should be discharged in a
Labeled Biohazard Bag Next to the Instrument.
Stem Cell Instrumentation Foundry
Hazard Waste
Hazardous Chemical List:
• Sodium Azide
-Beads
-Primary/2n d Abs
▫ All empty bottles should be discharged in a Labeled
Chemical Hazard Bag Next to the Instrument.
• Propidium Iodide
▫ Sample Tubes: should through in Labeled Biohazard
Bag or Container
▫ Pipette Tips: should through in Labeled Biohazard
Sharp Container
• Waste Sheath Fluid
▫ Should always be 10% Bleach (300-400ml bleach)
Stem Cell Instrumentation Foundry
Rules and regulations
Procedures and policies outlined in this
presentation are for the purpose of protecting
the SCIF wet laboratory workers and equipment.
Anyone found in violation of the aforementioned
procedures will suffer consequences, which may
include a loss of privileges or even a complete
loss of access to the SCIF. In order to prevent
such consequences, it is your responsibility to
review the SCIF SOPs.
Stem Cell Instrumentation Foundry
SCIF Flow Cytometry Additional
Rules
All users should:
• Complete the Orientation and pass the test
• Must Be trained, Certified, and signed off before
operating independently
• Required to signed the SCIF Disclosure Agreement
• Must follow the SCIF Flow Cytometry procedures
• Clean up the working area after finish
The first user of a day MUST empery the waste tank
The last user of a day MUST fill up the sheath tank
Stem Cell Instrumentation Foundry
SCIF Flow Cytometry Services
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The State of Art Machineries
All Aspects of Flow Cytometry Applications
Instruments Training
Assistance All First Time Users
Consulting on Projects
Data Interpretation
Setting up and Maintaining All SCIF Cytometers
Flow Cytometry Technical Support
Teaching Flow Cytometry