Materials

• Krumdieck slicer

• Incubator: 37^oC – 80% O₂ – 5% CO₂

• Surgery material (knife, scissor, forceps)

• 12 wells plate

• 1ml safelock tubes

• Liquid N₂

Solutions

• Culture medium: William E + Glutamax + Glucose (1.375gr/500mL William E medium) + Ciprofloxacin (2.5 ml/500 ml Williams E medium)

• Krebs solution: for 3L ( 300mL 10x Krebs + 6.30g NaHCO₃ + 13.50g Glucose + 7.14g HEPES + 2.7l cold ultra pure water)

• University of Wisconsin organ preservation solution (UW)

Protocol

1. Assemble the Krumdieck slicer. Turn on the cooler, connect the cooler tube with the slicer. Turn on the flow cabinet. Set the incubator to 37^oC – 80% O₂ – 5% CO₂

2. Prepare Krebs: Carbogenate Krebs for 20 minutes and adjust to pH 7.4 by NaOH 5M

3. Prepare medium: 1.3mL medium (WilliamE+Glucose+Ciprofloxacin) per well.

     Note: Medium must be filled into plates, oxygenated in incubator for 30min-1h before incubate with slice.

4. For each experiment condition: 3 slices for ATP, 3 slices for RNA, and 3 slices for histology

5. Pick up the kidney, store in ice-cold UW solution

6. Slicing

• Fill the Krumdieck slicer with Krebs

• Clean the kidney from excess tissue (eg. fat) surrounding the kidney with the surgical knife.

• Put the kidney into the holder place of Krumdieck slicer

• Turn on, and start slicing, adjust the thickness of the slice

• The thickness of the kidney slice is measured by wet weight (4-5 mg/slice)

8. Put 1 slice/well for incubation. Using thermal pad to keep the plate warm during handling.

9. Plate with slices is incubated in incubator while shaking at 90rpm.

10. Collecting sample (harvesting slice)

               for ATP (1 slice/1ml SONOP/ tube),

               for RNA (3 slices/ empty safelock tube)

               for histology (3 slices/4%Formalin well)

      ATP and RNA samples must be snap-frozen in liquid Nitrogen.

      3x1mL medium for each experiment condition will be collected into micronic-well.

11. Clean the slicer: Disassmble the slicer, put all parts in soap, wash carefully and transfer to water box. Rinse with Demiwater and 70% ethanol and allow them to dry.

Remarks: