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College of Life Sciences Nutrition, Dietetics, and Food Science
Tessem Laboratory - Diabetes Research Group

Specific Experiments

RXR α Protocol

Plating Cells:

  1. Aspirate media from a 100% confluent small flask of INS-1 832/13 cells
  2. Detach cells with 2 mL trypsin. Let sit in incubator for 5-10 mins (until all cells have detached from flask).
  3. Add 8 mL RPMI media.
  4. Mix thoroughly, and take out 0.5 mL from this mixture and put in an Eppendorf tube.
  5. Take out 100 μL from this tube and put in a new Eppendorf tube. Dilute with 900 μL 1x PBS (1:10 Dilution)
  6. Mix (use vortex) and take out 15 μL per side of the hemocytometer for cell counting.
  7. Add up the counts for both sides and divide by 10 (to get avg. # of cells per square). Then multiply this by 10 (to account for the 1:10 dilution). Then multiply by 10^4 to get cells per mL (this is telling us the number of cells per mL in the first eppendorf tube).
  8. Next, do (cells/well)/(cells/mL). Cells per well depends on how many you want. For this experiment, you want cells/well to be about 1.0x10^5. This formula will give you mL/well. So, if my formula gave me 65 uL per well and I was filling 48 wells, I could do (65 uL/well)x60 (which would account for two plates, or 48 wells, plus 12 extra wells just for safety) which would mean I would need 3.6 mL total. I would need to add 435 uL media per well to get to 0.5 mL per well, so I would need (435 uL/well)x60 =26.1 mL total media. I would take out 26.1 mL media and put it in a boat and take out 3.6 mL from the trypsinized flask and put it in the same boat fill the wells accordingly (0.5 mL per well) with the multi-channel pipette. Make sure to mix the cell mixture frequently using the multi-channel pipette.

Adding Virus – when plating two 24-well plates

  1. Do this at least 12 hours after plating. Make up a 5 uL virus + 4 mL media mix (if doing two 24 well plates). Mix the mixture aspirate the media on the first plate. After mixing, put 0.5 mL in each of the first 4 wells 2.
  2. Next, add 2 more mL media to the virus/media mixture and and mix again, and then put 0.5 mL of this mixture into the next four wells. Do this until you get to the control, where you will simply add 0.5 mL virus-free media per well. Repeat this process for the next plate.
  3. Leave virus on for 4 hours, then aspirate all wells and replace with 0.5 mL media per well.
  4. Let sit for about another 24 hours, then aspirate all media. Replace media from apoptosis plate with 0.5 mL 1.0 mM palmitate media per well, and the proliferation plate with 0.5 mL RPMI media per well. Let sit for another 24 hours (need to be pretty exact on this time), and then do cell counts. (In total, 48 hours after putting on virus).

Zinc Proliferation and Apoptosis

Day 0: Split and Plate cells

Materials:

  • Media
  • Trypsin
  • 10ml/5ml sterile pipettes
  • T-75 cell culture flask
  • Vacuum Apparatus
  • Bio Safety cabinet

Procedure:

  1. Prepare BioSafety cabinet.
    1. Turn off UV light
    2. Turn on venting and lighting
    3. Open Cabinet Door to Working Position
    4. Spray down inside surfaces of cabinet with 70% ethanol and wipe down
  2. Bring Materials and Reagents into Biosafety Cabinet.
    1. Spray with 70% ethanol and wipe down all surfaces before placing material or reagents into cabinet
    2. Use sterile reagents, filter non-sterile reagents if necessary
  3. Use vacuum Apparatus to aspirate off old media.
  4. Add 2ml Trypsin to the cell-containing surface.
  5. Let stand for 3-5 minutes, until all the cells are loosely floating (can be seen in microscope).
  6. Add 8ml media and mix until cells are consistently spread throughout the solution.
    1. Repeatedly suck suspension into the pipet then expel into flask, thus breaking up cell chains and groups.
  7. Pipet a calculated aliquot of cell suspension into the new unused cell culture flask.
    1. Q.S. to 25ml media and cells in new flask
    2. Mark new flask with cell line (823/13), split ratio, initials and date.
    3. Store flask in humidified incubator set to 37°C and 5% CO2.
  8. Count the cells in a small sample from the original flask. Maintain a ratio of 88 cells per 20ul of 50ul cell solution in 0.5ml of PBS.
    1. Correct by adding either more cells or more media below.
  9. Take 3/10 of a confluent flask per 24 well plate, so 6/10 confluent flask and place into a boat. Add 12 ml of media per plate (24ml total for 2 plates).
  10. Aspirate Unused cell solution and discard used flask.
  11. Clean up BioSafety Cabinet and return reagents/media to fridge.

Day 1: Add ZnCl2 to Plates

Materials:

  • 100mM ZnCl2
  • Media
  • 2 x 24 well plates from Day 0
  • 5ml sterile pipets
  • Empty troughs
  • Vacuum Apparatus
  • BioSafety Cabinet

Procedure:

  1. Ensure BioSafety Cabinet is Properly Prepared (See 1 and 2 above).
  2. Prepare 4mM solution of ZnCl2 from 100mM ZnCl2 Stock Solution in a trough.
    1. 40ul ZnCl2 + 1ml media = 4mM ZnCl2/media
    2. For 2-24 well plates, use 120ul ZnCl2 + 3ml media = 3ml 4mM ZnCl2/media
  3. Set up the plate so that 3 wells are each a different condition from 2mM to 0.03125mM and one control condition.
  4. Add 500ul of the 4mM ZnCl2/media to the side of each 2mM well and gently mix the media.
  5. Serial dilute 1:2, 500ul from each well down. Discard the waste before reaching the control wells.
  6. Gently mix the control wells with a new pipette tip.
  7. Discard waste with Vacuum.

Day 4: Plate Palmitate and New ZnCl2 to Apoptosis Plate and replate ZnCl2 and new media on the ZnCl2 Proliferation Plate

Materials:

  • 1.0mM Palmitate Media
  • ZnCl2 -RPMI Media
  • 24 well plates from Day1
  • Troughs
  • Sterile Pipettes
  • Vacuum Apparatus
  • BioSafety Cabinet

Procedure:

  1. Prepare BioSafety Cabinet according to Day 1 steps 1 and 2.
  2. Label one plate as ZnCl2 w/out Palmitate
    1. Aspirate off media from all wells.
    2. Prepare 4mM solution of ZnCl2 and RPMI media in trough.
      1. 80ul ZnCl2 + 2ml RPMI media = 2ml 4mM ZnCl2 Media.
    3. Add 0.5ml of media to side of each well.
    4. Add 0.5ml of ZnCl2 media to each of the 2mM wells.
      1. Mix well
    5. Serial Dilute 1:2, transfer 0.5ml to each well below, stopping at the control.
    6. Discard Waste and clean up Area.
  3. Label other plate as Palmitate.
    1. Aspirate off media from all wells.
    2. Add 0.5ml 1mM palmitate media to side of each well.
    3. Add 0.5 4mM ZnCl2 to the 2mM ZnCl2 wells
      1. Mix 80ul ZnCl2 and 2ml palmitate media in trough to create 4mM ZnCl2 Palmitate media. Add 0.5ml of this solution to each 2mM ZnCl2 well.
      2. Mix well
    4. Serial Dilute 1:2 to each well except the control wells. (Transfer 0.5ml to each of the following wells).

Day 5: Cell Counts

Materials:

  • Trypsin
  • PBS (not sterile)
  • Microscope
  • Cell counter
  • Hemocytometer
  • Kim Wipes

Procedure:

  1. Aspirate off old media.
  2. Add 100ul Trypsin to each well and allow to Trypsinize fully,
  3. Add 900ul PBS (not sterile) to each well.
  4. Count cells in a 20ul sample from each well and record.