1. Environmental Health and Safety
Standard Operating Procedure
BD FACSARIA III
Purpose
Test cell viability and determine whether the prototype cell collector is able to collect cells
Physical & Chemical Properties/Definition of Chemical Group
CAS#: not applicable
Class: not applicable
Molecular Formula: not applicable
Form (physical state): not applicable
Color: not applicable
Boiling point: not applicable
Potential Hazards/Toxicity
Disposing of cells into the appropriate biohazard container. As well as the potential to endanger ourselves
due to deflection plates that produce electricity.
Personal Protective Equipment (PPE)
Respiratory Protection not applicable
Respirators should be used only
under any of the following circumstances:
• As a last line of defense (i.e., after engineering and administrative controls have been
exhausted).
• When Permissible Exposure Limit (PEL) has exceeded or when there is a possibility that PEL will
be exceeded.
• Regulations require the use of a respirator.
• An employer requires the use of a respirator.
• There is potential for harmful exposure due to an atmospheric contaminant (in the absence of
PEL)
• As PPE in the event of a chemical spill clean-up process
Lab personnel intending to use/wear a respirator mask must be trained and fit-tested by EH&S. This is a
regulatory requirement.
Hand Protection
Latex gloves
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2. Environmental Health and Safety
NOTE: Consult with your preferred glove manufacturer to ensure that the gloves you plan on using are
compatible with cell collection
Refer to glove selection chart from the links below:
http://www.ansellpro.com/download/Ansell_8thEditionChemicalResistanceGuide.pdf
OR
http://www.allsafetyproducts.biz/page/74172
OR
http://www.showabestglove.com/site/default.aspx
OR
http://www.mapaglove.com/
Eye Protection
Safety glasses or chemical splash goggles. Goggles are preferred.
Skin and Body Protection
Lab personnel working with the chemical need to wear full-length pants or its equivalent, closed-toe
footwear with no skin being exposed, and a lab coat.
Hygiene Measures
Wash hands after working with the substance.
Engineering Controls
The tube which contains the cells will be placed in a compartment where the tube is held inside the
equipment (ARIA) which has the ability to close therefore there will not be any interaction between the
cells and the person’s conducting the experiment.
First Aid Procedures
If inhaled
Evacuate the victim to a safe area as soon as possible. Loosen tight clothing such as a collar, tie, belt or
waistband. If breathing is difficult, administer oxygen. If the victim is not breathing, perform mouth-to-
mouth resuscitation. WARNING: It may be hazardous to the person providing aid to give mouth-to-mouth
resuscitation when the inhaled material is toxic, infectious or corrosive. Seek immediate medical attention.
In case of skin contact
In case of contact, immediately flush skin with plenty of water for at least 15 minutes while removing
contaminated clothing and shoes. Cold water may be used. Wash clothing before reuse. Thoroughly
clean shoes before reuse. Get medical attention immediately.
In case of eye contact
Check for and remove any contact lenses. In case of contact, immediately flush eyes with plenty of water
for at least 15 minutes. Cold water may be used. Get medical attention.
If swallowed not applicable
Special Handling and Storage Requirements
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3. Environmental Health and Safety
In order to insure cell viability, cells are placed inside a refrigerator to keep cool and removed when
needed.
Spill and Accident Procedure
Chemical Spill Dial 9-911 and 228-7864
Spill – Assess the extent of danger. Help contaminated or injured persons. Evacuate the spill area.
Avoid breathing vapors. If possible, confine the spill to a small area using a spill kit or absorbent material.
Keep others from entering contaminated area (e.g., use caution tape, barriers, etc.).
Small (<1 L) – If you have training, you may assist in the clean-up effort. Use appropriate personal
protective equipment and clean-up material for chemical spilled. Double bag spill waste in clear plastic
bags, label and take to the next chemical waste pick-up.
Large (>1 L) – Dial 9-911 and EH&S at 228-7864 for assistance.
Chemical Spill on Body or Clothes – Remove clothing and rinse body thoroughly in emergency shower
for at least 15 minutes. Seek medical attention. Notify supervisor and EH&S at 228-7864 immediately.
Chemical Splash Into Eyes – Immediately rinse eyeball and inner surface of eyelid with water from the
emergency eyewash station for 15 minutes by forcibly holding the eye open. Seek medical attention.
Notify supervisor and EH&S at 228-7864 immediately.
Medical Emergency Dial 9-911 or 228-7864
Life Threatening Emergency, After Hours, Weekends And Holidays – Dial 9-911 Note: All serious
injuries must be reported to EH&S at 228-7864 within 8 hours.
Non-Life Threatening Emergency – Go to the Olivewood Meadows Occupational Health 374 Olive
during regular business hours. All other times report to Mercy Medical Center 315 Mercy Ave. Note: All
serious injuries must be reported to EH&S at 228-7864 within 8 hours.
Needle stick/puncture exposure (as applicable to chemical handling procedure) – Wash the affected
area with antiseptic soap and warm water for 15 minutes. For mucous membrane exposure, flush the
affected area for 15 minutes using an eyewash station. Go to the Olivewood Meadows Occupational
Health 374 Olive Ave. during regular business hours. All other times report to Mercy Medical Center 315
Mercy Ave. Note: All needle stick/puncture exposures must be reported to EH&S at 228-7864 within 8
hours.
Decontamination/Waste Disposal Procedure
Cells will be disposed of in the appropriate biohazard waste container as instructed by the Faculty
Supervisor.
General hazardous waste disposal guidelines:
Label Waste
• Affix an on-line hazardous waste tag on all waste containers using the Online Tag Program
https://ehs.ucop.edu/waste as soon as the first drop of waste is added to the container
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4. Environmental Health and Safety
Store Waste
• Store hazardous waste in closed containers, in secondary containment and in a designated
location
• Double-bag dry waste using transparent bags
• Waste must be under the control of the person generating & disposing of it
Dispose of Waste
• Dispose of regularly generated chemical waste within 90 days
• Call EH&S at 228-7864 for questions
• Empty Containers
o Dispose as hazardous waste if it once held extremely hazardous waste (irrespective of
the container size) A list can be found at
http://ehs.ucla.edu/Pub/ExtremelyHazardousWaste.pdf
Prepare for transport to pick-up location
 Check on-line waste tag
 Use secondary containment
Safety Data Sheet (SDS) Location
Online SDS can be accessed at http://ehs.ucmerced.edu/material-safety-data-sheets.
Protocol/Procedure
Start Up
Note. If you are the first user of the day, steps 1-9 will already be completed for you. If you need to
change nozzles or experience a major blockage, start from step 1.
1. Empty waste (A) down the lab sink if full. Decant 1L 70% Ethanol into the empty waste container.
2. Refill sheath tank (B) with 1x PBS to the top 'fill line' (Note. Use the autoclaved 1x PBS prepared
especially for this instrument. DO NOT use the NERL Diluent Sheath Fluid that is used for the
LSR2). Ensure air and fluid lines are connected to tank.
3. Switch on the computer and log in to Windows using the Admin account and password “BDIS”.
Wait for Windows to load until the network icon appears in the lower right system taskbar. Turn
on the compressed air line (orange valve on the wall). Then turn on the instrument (C), AMO (Aria
III only) and circulating water bath (4˚C, Aria III only).
4. Log in to windows (password BDIS). Open the program BD FACSDiva, log in using your
username and password. Wait for the cytometer to connect.
5. From the dropdown menu, select Cytometer>Fluidics startup, follow the prompts.
6. Note the nozzle size needed. Select the appropriate nozzle, and follow the steps in Installing the
Nozzle to ensure the configuration is correct.
7. Bleed sheath line filter (D) of air. Turn on the stream (red X on the stream window) (E). Allow 3-5
minutes for the stream to settle. Adjust the Amplitude so that the break-off point (S) is on screen
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5. Environmental Health and Safety
(Drop 1 should be ~100-300), and the Gap is stable: 70um ~6-10; 100um ~10-18 the frequency
node should be appropriate at the previously set frequency. If you want to inspect/change the
frequency node. A good check for stream stability is that side streams (F) are clean and stable.
8. Run CST
9. Turn the Sweet Spot (G) on
10. Set the drop delay
11. Set the side stream (F) trajectory
12. Run 10% bleach for 2 minutes, followed by DI H20 for 1 minute. Once you have finished your
sort, you should check the calendar booking system to see if you are the last user of the day. If
there is someone after you, complete the Final Clean. If you are the last user of the day, complete
the Shut Down procedure.
Final Clean
1. Run 10% bleach for 5 minutes
2. Run DI H20 for 5 minutes
3. Log out of your username in BD FACSDiva
Shut Down
1. Run 10% bleach for 5 minutes
2. Run DI H20 for 5 minutes
3. Turn the stream off (green tick on the Stream Window) (E)
4. Remove the nozzle and place it into its allocated 5ml tube containing fresh DI H20. Install the
closed loop nozzle. With ~4ml of DI H20 on the sample loading station (T),
selectCytometer>Cleaning Modes>Flow Cell Clean from the drop down menu. Repeat.
5. Export your data.
6. Quit BD FACSDiva, turn off the cytometer (C), computer and AMO. De-pressurise the sheath
tank (B).
Bulk sort
The collection apparatus will have a bulk sort into desired collection tube type of 50ml and two 5ml
collection tubes that can be housed simultaneously.
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6. Environmental Health and Safety
1. The amount of populations that will be collected will be noted and the collection housing will be
chosen accordingly.
2. The housing will be filled with blank dummy collection tubes and slide into place.
3. Press voltage (O) and test sort (P). Open the waste drawer (Q), and open the front of the sort
chamber.
4. Move the side stream charge sliders (R) out to a point where the side stream is targeted to the
bottom of its corresponding collection tube (verify this by looking down through the sort chamber
to the collection tubes below). Only set up those side streams you are planning to use.
5. UCM net ID will be used as the Diva username, a password must be made
6. If the Closed Loop Nozzle is in place, store it in the nozzle holder (H) to the right of the sort
chamber.
7. The values on the right side are the live values of the Gap/Drop 1.
8. The up/down arrows of the frequency field will adjust the frequency by 0.1 MHz
9. One node of potentially several
10. The bead lot is displayed on the bead vial
11. When the optical filter is applied, you will not be able to see the side stream. Turn the filter on and
off again to approximate the position of the side stream within it.
12. If you cannot attain 100, try adjusting the Accudrop laser.
13. For most 1 or 2-way applications, this will be purity. For most 3 or 4-way applications, this will be
4-way.
14. The goal here is to accurately find the point where the most beads (highest left side stream
number) are deflected. This implies that the drop delay reflects the time is takes for a segment of
stream to move from the laser interrogation point to the droplet break-off (S).
NOTE
Any deviation from this SOP requires approval from PI.
Documentation of Training
• Prior to conducting any work with BD FACSARIA III, designated personnel must provide training to
his/her laboratory personnel specific to the hazards involved in working with this substance, work
area decontamination, and emergency procedures.
• The Principal Investigator must provide his/her laboratory personnel with a copy of this SOP and a
copy of the SDS provided by the manufacturer.
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7. Environmental Health and Safety
• The Principal Investigator must ensure that his/her laboratory personnel have attended appropriate
laboratory safety training or refresher training within the last one year.
I have read and understand the content, requirements, and responsibilities of this SOP:
Name Signature Date
.
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