Data Parameters and Units:
% Tail Intensity (reflects % DNA in tail) Wetzel_comet_data.xlsx-- Sample Info: MML #, Sample ID, Catch ID, matrix (liver, muscle, blood, placenta, whole pup, gonads, kidney, serum), analysis (PAH, comet, fertility, immune), Set ID, Collection Date (MM/DD/YYYY), Time (HH:MM), Common name (Silky, Shortfin Mako, Wahoo, Scalloped hammerhead, Great hammerhead, Oilfish, Pelagic stingray, Lancetfish, Yellowfin tuna, Escolar, Mahi Mahi, Swordfish, Sandbar, Spinner, Blacktip, Baracuda, Dusky, Bull, Blacknose, Tiger shark), Species, Sex (Male, Female, pup), PCL Precaudal Length (cm), FL Fork Length(cm), TL Total Length(cm), STL Stretch Total Length (cm), Girth, Weight(kg), Liver wt (kg), Maturity (Mature, Immature, pup), Comments. Comet Data: % Tail Intensity and % Tail Moment for: Source CC0.xls, Source CC0.xls, Source CC1.xls, Source CC1.xls, Source CC2.xls, Source CC2.xls, Source CC3.xls, Source CC3.xls, Source DU001, Source DU002, Source SI003, Source SI004, Source SB003, Source SB0001, Source SB0009, Source SB0004-06, Source SB0006-06, Source SM001, Source SP0001-06, Source SI001, Source SB00010, Source SB0002-06, Source SB0007-06, Source BT0001, Source BG-2, Source BG-5, Source BG-7, Source BG-8, Source SH-001, Source SW-031, Source BG-1 (BN). Comet Graphs: Sample IDs verses % Tail Intensity and Sample IDs verses % Tail Moment.
Methods:
Sample Collection Methods: - Collect a quarter size portion of each tissue into a scintillation vial and store on ice. - Wash 3 times with approximately 2 mL L-15 media. Discard each wash. - Add 2 mL of 4C Tissue Preparation solution and mince tissue into 1 mm pieces - Incubate on ice for 5 minutes - Liver – after 5 minute incubation, pass sample through 40 micron filter, stacked atop a 50 mL conical vial. Scrape tissue until all supernatant runs through. Additional PBS can be used, if needed. - Collect supernatant into a yellow capped centrifuge tube and centrifuge at 1200 rpm for 10 minutes. - Aspirate and discard supernatant and resuspend cell pellet in 1 mL 4C PBS - Count cells and check viability using a 1:1 dilution of Trypan blue with 10 uL cell suspension. - Adjust volume of PBS to the suspension to yield 1x105 cells/mL - Centrifuge at 1200 rpm for 10 minutes, aspirate and discard supernatant, and resuspend in an equivalent amount of Cryopreservation media. - Place cryovials in cool cell overnight. In the morning, transfer to LN2. Sample Preparation in the laboratory: I. Solution Preparation A. Alkaline Electrophoresis Solution pH >13 (200 mM NaOH, 1mM EDTA) i. 8g Sodium Hydroxide (NaOH) ii. 2 mL 500 mM EDTA, pH 8 iii. Bring to 1000 mL with DI water (after NaOH is dissolved) iv. Chill at 4 ° C B. Lysis Solution i. Chill bottle at 4 ° C for at least 20 minutes C. Alkaline Solution i. 0.6g Sodium Hydroxide (NaOH) ii. 250 µl 200mM EDTA iii. 49.75 mL DI water iv. Allow to cool to room temperature II. Electrophoresis Unit Preparation A. Set up electrophoresis unit in the cold room, with alkaline electrophoresis solution i. 300 mA, constant amp, 21 V ii. Make sure unit is level iii. Clean jack ports with isopropyl alcohol III. LMAgarose Preparation A. Melt agarose by placing bottle in boiling water for 5 minutes. B. Aliquot enough agarose for Comet slides (500 µl per slide) into eppendorf tubes and float in 37°C water bath for a minimum of 20 minutes to cool. IV. Comet Slide Preparation A. Place appropriate number of comet slides in 37°C oven. V. Protocol 3. Thaw cell suspensions quickly to room temperature by submerging in 37°C water bath then placing on ice. 4. Centrifuge cells at 150 x g for 5 minutes and gently remove the supernatant. 5. Resuspend in ice cold PBS using the same volume the cells were preserved in. 6. Add 50 µl of 1 x 105 cells/mL cell suspension to 500ul 37°C LMAgarose 7. Gently mix by pipetting up and down. 8. Transfer 50µl to sample area on comet slide. Use pipette tip to spread mixture over entire sample area. 9. Repeat steps 2-4 for each sample. 10. Lay slides flat in a container with a small amount of desiccant and place at 4°C for 30 minutes. 11. Immerse slides in prechilled lysis solution and leave at 4°C for 30 minutes. 12. Drain excess lysis solution from slides and place slides in room temperature alkaline solution 13. Let stand in the dark for 20 minutes 14. Add 950 mL Alkaline Electrophoresis Solution to electrophoresis chamber 15. Place slides in electrophoresis unit slide tray a. Slide label on the side of the black cathode, samples towards the red anode b. 2 slides per slot 16. Cover slides with slide tray overlay 17. Place lid on electrophoresis unit ensuring proper connection of wires 18. Set power supply to constant amp, 300 mA, 21 volts; 30 minutes 19. Drain excess liquid form slides, immerse in DI water, incubate for 10 minutes 20. Repeat step 19 one time 21. Drain excess liquid form slides and immerse in 70% Ethanol 22. Incubate for 5 minutes 23. Dry samples for 10-15 at room temperature Samples may be stored at room temp with desiccant before scoring at this stage 24. Dilute SYBR green II (stable once prepared for several weeks when stored at 4°C in the dark) a. 1µl SYBR green b. 10mL TE buffer, pH 7.5 25. Place 100ul of diluted SYBR green onto each sample area and incubate at 4°C for 5 minutes 26. Gently tap slide to remove excess liquid 27. Dry at room temperature in the dark 28. View and score slides by epifluorescence microscope.
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