Fluorogenic Tissue-Based Assessment of Acid Ceramidase Activity

Materials and reagents

Biological materials

1. GBA1^+/+ mouse brain tissue (WT mice, offspring of GBA1^+/L444P heterozygotes originally obtained from the University of North Carolina at Chapel Hill Mutant Mouse Resource and Research Center, catalog number/origin: MMRRC-000117)

Reagents

1. Dimethyl sulfoxide (DMSO) (Fisher Scientific, catalog number: D12345)

2. D-Sucrose (Fisher Scientific, catalog number: BP220-10)

3. EDTA (0.5 M, pH 8.0) (Fisher Scientific, catalog number: 15575020)

4. Glycine (Fisher Scientific, catalog number: BP381-500)

5. MeOH (Fisher Scientific, catalog number: A411-4)

6. Sodium periodate (NaIO₄) (Millipore Sigma, catalog number: 311448-5G)

7. Pierce BCA Protein Assay kit (Fisher Scientific, catalog number: 23225)

8. Rbm14-12 substrate (Avanti Polar Lipids, catalog number: A86855)

9. Sodium acetate (1 M, pH 4.5) (Fisher Scientific, catalog number: J63669.AE)

10. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-212)

11. Sodium hydroxide (NaOH) (1 M) (Fisher Scientific, catalog number: SS266-1)

12. Tris base (Fisher Scientific, catalog number: BP152-500)

13. Umbelliferone (Millipore Sigma, catalog number: H24003)

Solutions

1. Assay solution (see Recipes)

2. Detergent-free lysis buffer (see Recipes)

3. 100 mM glycine/NaOH buffer (see Recipes)

4. 4 mM Rbm14-12 substrate stock solution (see Recipes)

5. 25 mM Sodium acetate buffer (see Recipes)

6. 2.5 mg/mL NaIO₄ solution (see Recipes)

7. 0.2 M sucrose solution (see Recipes)

8. 5 mM umbelliferone stock solution (see Recipes)

Recipes

1. Assay solution (per well)

*Conditions are to be optimized by the experimenter based on tissue type and animal model. See the aCDase activity assay reagent calculator in Supplementary information, Template 1, to easily calculate the amount of 4 mM Rbm14-12 substrate and the volume of 25 mM sodium acetate required to prepare the desired substrate concentration. For example, for a desired Rbm14-12 substrate concentration of 5 μM, 0.125 μL of 4 mM Rbm14-12 stock solution would be required per 100 μL of total volume. This can be scaled up using the aCDase activity assay reagent calculator in Template 1, so that a multi-channel pipette can be used to add 75 μL of assay solution to each necessary well.

The assay solution should be made fresh the day of the experiment.

2. Detergent-free lysis buffer

The detergent-free lysis buffer can be stored at room temperature (20–25 °C) for long-term storage. Collected protein supernatants using the detergent-free lysis buffer can be stored at -80 °C for long-term storage. Please note that protease and phosphatase inhibitors are omitted from the lysis buffer. To prevent any changes in enzyme stability, it is recommended to use chilled buffers, keep samples on ice, and work as quickly as possible while maintaining accuracy.

3. 100 mM glycine/NaOH buffer

The 100 mM glycine/NaOH buffer should be stored at 4 °C for up to two weeks, as long as the pH remains stable and unchanged. Please note that it is recommended to add 1.875 g of glycine to 200 mL of MilliQ water. Adjust the pH to 10.6 by slowly adding 1 M NaOH while stirring. After the solution reaches a pH of 10.6 based on the pH meter, add enough MilliQ water to bring the final volume to 250 mL. Mix thoroughly and verify the pH, adjusting if necessary.

4. 4 mM Rbm14-12 substrate stock solution

Due to the volatility of ethanol (EtOH), it is recommended to dissolve Rbm14-12 in DMSO, rather than EtOH. The Rbm14-12 substrate stock solution in DMSO (4 mM) should be stored at -20 °C for <1 month in an amber glass vial wrapped in foil. It is recommended to aliquot the DMSO stock solutions of Rbm14-12 prior to storage to minimize freeze-thaw cycles.

5. 25 mM sodium acetate buffer

The 25 mM sodium acetate buffer can either be stored at 4 °C or room temperature (20–25 °C) for up to two weeks, as long as the pH (4.5) remains stable and unchanged.

6. 2.5 mg/mL NaIO₄ solution

The 2.5 mg/mL NaIO₄ solution should be made fresh the day of the experiment.

7. 0.2 M sucrose solution

The 0.2 M sucrose solution can be stored at 4 °C for up to two weeks, as long as the pH (6.0–6.5) remains stable and unchanged. Please note: to prepare the 1 M sucrose used for the final 0.2 M sucrose solution, 17.12 g of sucrose is dissolved in 250 mL of MilliQ water. The remaining 1 M sucrose solution that is not used can either be discarded or stored at 4 °C for up to two weeks.

8. 5 mM umbelliferone stock solution

Due to the volatility of EtOH, it is recommended to dissolve umbelliferone in DMSO, rather than EtOH. The umbelliferone stock solution in DMSO (5 mM) should be stored at -20 °C for long-term storage. It is recommended to aliquot the DMSO stock solutions of umbelliferone prior to storage at -20 °C to minimize freeze-thaw cycles.

Laboratory supplies

1. Amber glass vial (Millipore Sigma, catalog number: 27046-U)

2. Black, flat-bottomed 96-well plate (Fisher Scientific, catalog number: M33090)

3. Microcentrifuge tubes (1.5 mL) (Fisher Scientific, catalog number: 05-408-130)

4. Microcentrifuge tubes (0.6 mL) (Fisher Scientific, catalog number: 05-408-120)

5. Motorized tissue grinder (Fisher Scientific, catalog number: 12-141-361)

6. Pellet pestles (Fisher Scientific, catalog number: 749521-1500)

Equipment

1. Microcentrifuge (Fisher Scientific, catalog number: 75002446, model: Sorvall Legend Micro 21R)

2. Microplate reader for BCA (Agilent Technologies, catalog number: 209924, model: BioTek Synergy 2 SL)

3. Microplate reader for umbelliferone detection (Molecular Devices, model: SpectraMax iD3)

Procedure