Stem Cell Instrumentation Foundry SCIF Orientation Flow Cytometry Venu Polineni Research Associate, Stem Cell Instrumentation Foundry UCM 5200 N. Lake.

Stem Cell Instrumentation Foundry

SCIF Orientation Flow Cytometry

Venu Polineni Research Associate,

Stem Cell Instrumentation FoundryUCM 5200 N. Lake Road Merced, CA

95343 Email: [email protected]

mailto:[email protected]


Overview of Flow Cytometry

Part I: Cell Preparation• 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• 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


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


SCIF Flow Cytometry Instrumentation

BD LSRII Multi-Laser Analyzer BD AriaII/III Multi-Laser Sorter

http://www.productwiki.com/upload/images/becton_dickinson_lsrii.jpg



LSRII: Analyzer

Aria II: Cell Sorter



Aria III Cell Sorter


Safety

•-General Lab Safety

•-Electrical Safety•-Optical/Laser Safety

•-Biohazard Safety


Symbols

Electrical Shock Laser Irradiation BiohazardCaution

• 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


General Lab SafetyThe 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


Electrical Safety

• Stream ChargeWhen 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 VoltageThis 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.


Electrical Charge of Sorting


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.


Biological Effects of Laser Irradiation

•Eye Injuryeye 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 (315-390 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).


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.


Biohazard Safety

IMPORTANTIf 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.


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.


Hazard WasteHazardous 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)


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.


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


SCIF Flow Cytometry Services

•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

Stem Cell Instrumentation Foundry SCIF Orientation Flow Cytometry Venu Polineni Research Associate, Stem Cell Instrumentation Foundry UCM 5200 N. Lake.

Documents

cell preparation

cell sorter slide

specific cell population

instrumentation sample

flow software

flow core

live pi budr ao cell

virus slide