Microsoft word - pgb003 tunel assay

Protocol Title
TUNEL Assay (Transwell)
Protocol Number
Dates Modified
03 Sept 2010

METHOD
Indicate detailed steps of method.
Use SI units for quantities, xg plus rpm for named centrifuges, and indicate starting and final
concentrations for use of reagents.

Equipment
Pipettes, Pipette Tips, Waste container, Liquid Waste Container, Lab coat,
Gloves, Racks for 50ml and 15ml tubes, Paper towels, 70% Alcohol spray
bottle, Presept tablets, 15 ml BD conical tubes, 50ml BD Falcon tubes,
Corning Polycarbonate Transwell Supports (0.4m pore size, 6.5mm Order
No.), Diff-Quik stain set, forceps, glass slides, round coverslips, curved
scalpel, eppendorf tubes, 24 well plates or 96 well plates, foil, fluorescent
microscope.
Solutions and reagents
Sterile PBS without calcium and magnesium
Vectashield Hardset mounting medium with DAPI
TUNEL stain set
Neutral buffered formalin (3.7%)
Cell culture medium
Protocol
Safety considerations:
Lab coats and gloves must be worn.
Formalin/Farmaldehyde is extremely toxic and must be handled in fume
cupboard.
General points:
All procedures are at room temperature unless otherwise stated.
Use BD tubes not Greiner.
Controls for this experiment should include:
Negative – untreated cells labeled with labeling solution + enzyme
Negative – untreated cells incubated with labeling solution without enzyme
Positive – cells treated with an apoptosis inducer such as etoposide,
doxorubicin, gliotoxin, LL-37 etc.
For Cultures on Transwells
1) In a tissue culture cabinet and with sterile gloves, remove required number
of transwells from packaging and transfer to a new 24 well plate. Add 600l of pre-warmed culture medium to the bottom of each well and then place the transwell on top, taking care to ensure that there are no air bubbles below the membrane. Place in a tissue culture incubator for 15 -20 minutes. 2) If using epithelial cells, trypsinise the cells and adjust cell number to 300,000 / ml in normal culture medium. Then remove the transwells from the incubator and, in a culture hood, add 100l of cell suspension (i.e. 30,000 cells) to each transwell and allow to stick down over night in a 37oC incubator. 3) The following day, prepare the treatments in 700l culture medium (with or without serum). Then, when required, remove the media from above and below the transwells and replace with media containing treatment compound. A washing step with sterile PBS can be inserted here if necessary. Then incubate cells with relevant treatments. 4) Following treatment, remove the media containing the treatment substance from the transwells. Note: If required, 80l of the media from above the transwell membrane can be utilized for a cytospin. This is especially recommended for high toxicity treatments which may cause cells to detach from the polycarbonate transwell membrane. The media from the lower chamber can also be frozen for future analysis. Invert each transwell on a piece of tissue paper and allow to air dry for 45 minutes at room temperature. 5) Replace transwells in a clean 24 well plate and add 200l of neutral buffered formalin (3.7%) to the top of each transwell. Incubate at room temperature for 10 minutes. 6) Aspirate the formalin from each well and wash once with PBS. Then add 200l of ice cold cell permeabilisation solution (0.1% Triton X100 / 0.1% sodium citrate) to each transwell and incubate at room temperature for 3 minutes. Remove the solution from the wells and wash twice with PBS. 7) Now to label the cells using the Roche In Situ Cell Death Kit. Remove 50l from the labeling solution and store in a separate tube for an enzyme control. Then add 50l of enzyme solution to the labeling solution (total volume now 500l). 8) Remove any PBS from the transwells and add 75l of labeling solution to each well and incubate at 37°C in the dark for 1 hour. Remember to add the enzyme control to one well. Note: If required, you can economise on the labeling solution to the extent that the minimum volume that can be added to each well can be approximately 40-45l. 9) Following labeling, remove the solution from the wells and wash three times with PBS. Carefully touch the bottom of the transwell to tissue paper to remove any excess liquid. Then use a curved scalpel and forceps to remove the polycarbonate membrane from the transwell and place cell-side up on a glass slide. Add 10l of Vectashield Hardset with DAPI to the top of the filter and cover with a round glass coverslip. Allow to set for 1 hour in the cold room wrapped in foil. 10) Using the Axiovert 5100 microscope, count at least 300 cells in five fields of view. Apoptotic cells will be labeled bright green. 1. Seed 20-30,000 cells per well and incubate plate at 37oC + 5% CO2 2. Remove media and wash cells with warmed sterile PBS 3. Treat cells using treatment media containing DMEM w/o phenol red, L- glut, NEAAs, UltroserG (if serum substitute required) and treatment of interest. 4. Following treatment remove the culture media and invert plate allowing 5. Fix and permeabolise the cells by adding ice cold acetone:methanol 6. Wash using sterile PBS 7. Label the cells using the Roche In Situ Cell Death Kit. Remove 50l from the labeling solution and store in a separate tube for an enzyme control. Then add 50l of enzyme solution to the labeling solution (total volume now 500l). 8. Remove any PBS from the wells and add a minimum of 45l of labeling solution to each well (though more is desirable) and incubate at 37°C in the dark for 1 hour. Remember to add the enzyme control to one well. 9. Following labeling, remove the solution from the wells and wash three times with PBS. For second PBS wash include DAPI at 1 g/ml. 10. Using the Axiovert 5100 microscope, count at least 300 cells in five fields of view. Apoptotic cells will be labeled bright green. SOLUTION DETAILS
Use full names and chemical formulae, use SI units for quantities, record MW of chemicals used,
show molarity and wt/vol of solutions, record storage details and any other special requirements

Neutral buffered formalin (3.7%)
Formalin / formaldehyde is stored at 37% in the poisons cupboard. Add 1ml of
formalin to 9ml of PBS.
Cell Permeabilisation Solution (0.1% Triton X-100 / 0.1% Sodium citrate)
Triton X-100 is stored on the general chemical shelf at 100%. Sodium citrate
is stored in the tissue culture room with the blood prep equipment at 3.8%. To
prepare 10ml of solution, add 10l of Triton X-100 and 263l of 3.8% sodium
citrate to 10ml distilled water.
Vectashield Hardset with DAPI
The Vectashield is stored in the cold room at 4oC. The DAPI is stored at -20oC
in either dessicated form or in glycerol. If dessicated, make a suspension of
DAPI in 50% glycerol at a stock concentration of 1mg/ml. Then add 1l of
DAPI suspension to 1ml of Vectashield to a final concentration of 1l/ml.
SOURCE OF REAGENTS
Record supplier, catalog number, method to make ready for us, storage site, any special storage
requirements
REFERENCES
Indicate any literature source for the method

Authors, (Year), Journal, Volume, Page numbers
MODIFICATIONS
When any modification is made to this method, record the change here to keep a record of the
old methodology with the date change made recorded here and top of protocol.
Modification 1 Date: Changes: Modification 2 Date: Changes:

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