Protocols_PWS CS_Liquid SlidePrep_V1 - BackmanLab/BackmanLab.github.io GitHub Wiki

+----------------------+-------------------------------------+ | Procedure: | Clinical Sample -- Liquid | | | | | | Cell Isolation for PWS Analysis | +======================+=====================================+ | SOP Prepared by: | Jianan Lin & Adam Horton | +----------------------+-------------------------------------+ | Version: | 1.0 | +----------------------+-------------------------------------+

1. Scope

The purpose of this SOP is to describe how to prepare human samples for PWS analysis.

2. Description

This protocol aims to describe the procedure for in vivo sample collection and treatment for posterior analysis by partial wave spectroscopy (PWS). Samples can only be collected after IRB approval of the collection protocol. Sample collection can only be performed by a board certified expert endoscopist after a written consent was obtained.

3. Personal Protective Equipment

• Lab coat

• Gloves

• Mask (located

4. Potential Hazards + Safety Precautions

• Lab coat, and nitrile gloves should be used at all times.

• Sample deposition on slide: Although the risk is minimal, spray of a clinical sample may cause hazardous aerosols. Handle in the fume hood and use a mask.

5. Materials and Reagents

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1. Equipment

• Thermal Label maker (Brady, MB51(53), in the pedestal under shared

  desk in corridor B-741)

• Spray deposition compressor (on the bench by the fume hood in B-715)

• Spray gun

• Inverted microscope (Room# B-715 shared bench)

• Refrigerated centrifuge (Eppendorf 5418R, tissue culture room

  B-749).

2. Materials

• Pipettes and tips

= Clinical Sample -- Liquid Cell Isolation for PWS Analysis (Cont.) =

• Glass Slides (*Fisherbrand, Superfrost Microscope Slides,

  Precleaned, Cat# 12-550-143, 25 x 75 x 1.0 mm* - located on the drawer under the common bench in room B-715)

• Mask (3M, Particulate Respirator N95, Cat# 8210)

• 1L Glass beaker

• Absorbent paper towels

• Slide Labels (Brady, Microscopy slide labels, Cat # M-194-481)

• Tube holder

• Forceps

• Spray gun

• Slide box

• Waste container for chemically contaminated plastic sharps (e.g.

  pipette tips).

3. Reagents and Solutions

• High Purity Denatured EtOH (Thermo Sci., , Ref# 9200-1)

• RO water (tap on the lab sinks)

• 25% Ethanol (EtOH):

  • 25 mL denatured EtOH + 75 mL of RO water (RO tap on the lab sink)

• 95% Ethanol:

  • 95 mL denatured Ethanol + 5 mL of RO water

• 70% Ethanol:

  • 70 mL denatured Ethanol + 30 mL of RO water

= Clinical Sample -- Liquid Cell Isolation for PWS Analysis (Cont.) =

6. Procedure

Sample Reception:

Samples are shipped same day (local) or overnight (out of town) by the clinical sites.

Samples are shipped in a biohazard bag, refrigerated (at 4^o^C) in an insulated bag within a polystyrene box.

Samples can be picked up 8:00 a.m. - 5:00 p.m., Monday to Friday from the mailroom in the Tech building, Room MG92 (Phone: 847-491-5424). Alternatively, samples will be delivered to the lab after lunch by the mailroom staff.

Sample Control:

Once packages arrive check overall package condition for any abnormalities.

1. In your Lab book, annotate:

• Ice pack status (e.g. frozen | partially frozen | melted).

• Vial solution expiring date.

• Sample status (e.g. color; turbidity; presence of debris; saturation

  level; etc.)

• Any other relevant information.

2. Keep the sample at 4^o^C while preparing all the required materials

   and solutions.

3. Label each of your slide(s). Make sure to include:

   • Study ID

   • Sample ID#

   • Date of preparation

   • Preparer name/initials

   • Replicate # (if more than one)

NOTE 1: There are Cryo-tube labels (Ref# M-122-461) available to be used with the Thermal printer (see Thermal Printer User Guide for more information). This labels don't fade, have a transparent part that will go over the writable section for extra protection and are designed to withstand ultra-low temperatures without peeling off from the tube.

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= Clinical Sample -- Liquid Cell Isolation for PWS Analysis (Cont.) =

{width="1.886111111111111in" height="3.1226410761154857in"}Slide Preparation - Liquid Airspray Deposition:

1. Place several paper towels in the bottom of the 1L beaker located in

   the fume hood to catch the aerosols during the spray gun cleaning process.

2. Turn on the deposition gun switch (a), turn the pressure to between

   20-25 psi on knob (b).

3. Move the spray gun (c) onto the fumehood

4. Place a clean absorbing towel in the fume hood base for

5. Clean the deposition gun

   • Clean 3x with 1 mL distilled water using a 1 mL automatic

     pipette. Insert the pipette tip in the top opening of the spray gun (c) and dispense the liquid.

NOTE 2: Make sure not to touch the walls or bottom of the gun.

• Spray the gun into the beaker of paper towels until there is no more

  liquid visible out of the spray gun.

• Make sure that the pressure is at 20 Psi when the gun is spraying.

• Repeat the above procedure using 95% EtOH.

6. Cell deposition

6.1. Cover the fume hood surface where you'll be spraying the slide with absorbing paper towels.

6.2. Clean the exterior of the automatic pipette with 70% EtOH using a paper towel.

6.3. Remove the brush from the sample collection tube using tongs and place in the collection biohazard bag.

6.4. Pipette up and down to mix the sample

6.5. Using the 200 mL Pipette, pipette 150 µL of sample onto the deposition gun.

6.6. Use the marked pole to make sure you hold the gun vertically 6 inches above the slide.

6.7. Depress the trigger to the soft stop (no sample should come out), then move the gun directly over the slide

6.8. Deposit the sample onto the slide moving the gun gently back and forth to cover the entire slide surface.

6.9. Remove the slide from the fume hood and allow it to air-dry on the bench.

NOTE 3: Make sure you place your slides over a disposable paper towel

= Clinical Sample -- Liquid Cell Isolation for PWS Analysis (Cont.) =

6.10. Repeat steps 6.4. to 6.9. for each of your replicates.

6.11. Cap the biological sample tube and store in the fridge (4^o^C).

6.12. Discard the contaminated paper towels into the chemical waste bin.

6.13. Clean all reusable materials (pipettes, tube holders, tongs, etc.) with 75% EtOH and place them back in their original location.

6.14. If the waste container is full, seal it with a waste label and place it in in the gray chemical waste bin (located in the corridor by Room B-743's entrance).

7. Cell fixation

7.1. Refresh the 95% EtOH used for fixing the cells (located in a labeled bottle under the fume hood).

7.2. Once the deposited cells are dry place them into the 95% EtOH for 15 minutes in order to fix them.

7.3. Remove slides from EtOH and allow them to air dry on the bench (see NOTE 2 above).

8. Slide/Cells quality control

• Visualize the slides under the microscope to determine cell quality.

  A good quality slide should have at least the following characteristics:

  • ≥ 30 isolated cells (e.g. colonocytes for rectal brushings or

    buccal cells for oral brushings, etc.) either in a single slide or combination of the several replicates.

  • Cells should be intact.

  • Cells should be clean and well isolated

INSERT SLIDES IMAGES HERE

• Store slides at 4^o^C until PWS analysis.

NOTE 4: If the cells are not up to a workable quality (i.e., there is a lot of debris or fecal contamination visible), follow the centrifugation protocol below followed by steps 6. to 8. above (deposition process) on new slides.

Centrifugation

= Clinical Sample -- Liquid Cell Isolation for PWS Analysis (Cont.) =

1. Refrigerate the centrifuge down to 4^o^C

2. Fill up to the 1.5 mL mark of the centrifuge tube.

3. Pellet the cells at 400 x g, 4°C for 20 minutes using the Eppendorf

   centrifuge.

NOTE 5: Make sure the centrifuge is balanced and at 4^o^C before start.

4. Remove the supernatant using a pipette.

5. Resuspend the pellet in 1-1.5mL of 25% EtOH.

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7. Disposals | Spills | Incidents

Waste (see waste segregations and collection SOP for complete information)

• All waste containers should be closed once ¾ 's and placed into the appropriate location for pick up full.

• All waste containers should be properly labelled with the following information:

  • PI's name

  • User | Responsible person

  • Content

  • Waste class (e.g. chemical, biohazard, radioactive, etc.)

  • Waste type (e.g. sharps, plastic, glass, metal, etc.)

  • Waste "status" (e.g. liquid, solid, etc.)

[Protocol Specific]{.ul}

1. Waste

   • Plastic sharps -- place in a puncture resistant container with a

     "Chemically contaminated waste" labelled (e.g. plastic sharps/tips; )

   • Biological and chemical liquid spray -- use absorbing paper

     towels under the area around the slide. Discarded them as chemical waste in the plastic transparent bags.

   • Glass slides -- discard in a "glass card box".

   • Empty bottles of EtOH -- remove or unutilize the label, and mark

     as "rinsed to dispose".

     • Rinse 3x, leave open in the fume hood to dry;

     • Place by/in the regular waste bins for collection with

       regular waste.

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   • Biological samples -- inactivate with 10% bleach for 30 minutes and discard in lab sink.

2. Spills | Incidents:

Considering the type of reagents used (EtOH) and sample's volume (less than 5 mL), the risk of hazardous spills is minimal to non-existent.

= Clinical Sample -- Liquid Cell Isolation for PWS Analysis (Cont.) =

However in the event of an incident with a biological sample, make sure you use a mask, gloves and lab coat prior to spray the area with 75% EtOH or 10% bleach. Discard the absorbing towels as chemical waste.

8. References

= Clinical Sample -- Liquid Cell Isolation for PWS Analysis (Cont.) =

SUMMARY FLOWCHART OF PROCEDURES TO CONSIDER IN CASE OF A

xxxx EMERGENCY

= Clinical Sample -- Liquid Cell Isolation for PWS Analysis (Cont.) =

// Protocol Summary//

NOTE: This protocol summary should be [used only]{.ul} as "side bench quick reference" and only once you're fully familiar with the full protocol above, including hazards and safety measures.

Slide Preparation - Liquid Airspray Deposition:

1. Place several paper towels in the bottom of the 1L beaker and the

   fume hood base.

2. Turn on the deposition gun switch (a), turn the pressure to between

   20-25 psi on knob (b).

3. Clean 3x the deposition gun 1 mL distilled water

NOTE 2: Make sure not to touch the walls or bottom of the gun.

• Spray the gun into the beaker of paper towels until there is no more

  liquid visible.

• Make sure that the pressure is at 20 Psi when the gun is spraying.

• Repeat the above procedure using 95% EtOH.

4. Cell deposition

4.1. Clean the pipette with 70% EtOH

4.2. Remove the brush and pipette up and down to mix the sample.

4.3. Pipette 150 µL of sample onto the deposition gun.

4.4. Deposit the sample onto the slide.

4.5. Air-dry slide on the bench.

NOTE 3: Make sure you place your slides over a disposable paper towel

4.6. Repeat steps 6.4. to 6.9. for each of your replicates.

5. Cell fixation

5.1. Fix the cells into the 95% EtOH for 15 minutes.

5.2. Air dry slides on the bench.

6. Check Slide/Cells quality control

7. Store at 4^o^C until analysis