Endocannabinoid Uptake Studies In Vitro Assay of Endocannabinoid Uptake

1. COS cells (107) grown to confluence in flasks in DMEM (Life Technologies, Gaithersburg, MD) containing 10% FCS (Life Technologies) were split 1:2, harvested the next day using trypsin/EDTA, centrifuged (200g) for 10 min at 4°C, washed with sterile HEBS (20 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, and 6 mM dextrose), recentrifuged, and resuspended at 107 cells/mL in 4°C HEBS. Then, 0.9 mL of suspended cells was transfectedby electroporation at 300 V and 1100 |F in 400-mm Gene Pulser cuvettes (Bio-Rad) containing 20 |g of plasmid DNA and 500 |g of fish sperm DNA (Boehringer-Mannheim, Mannheim, Germany) using a geneZAPPER 450/2500 (IBI, New Haven, CT).

2. The transfected cells were suspended in DMEM, followed by distribution into 6-well plates. Cells were grown for 3 d and then assayed for their abilities to accumulate tritium-labeled anandamide [arachidonyl-5,6,8,9,11,12,14,15-3H]-, 160 Ci/mmol (New England Nuclear Research Products, Boston, MA).

3. Kinetic and saturation analyses were used to determine Km, Vmax values. For uptake assays, 1 nM tritium-labeled anandamide [arachidonyl-5,6,8,9,11,12,14,15-3H]-, 25 |Ci (925 kBq), and 0.1, 1, 2, 4, 10, and 20 |M unlabeled anandamide concentrations were used.

4. COS cells transfected with pcDNA3.1/V7-3 and the vector pCDNA3.1 served as positive and negative controls, respectively. Parallel incubations with 30 | M of unlabeled anandamide allowed estimation of nonspecific uptake and binding. Uptake assays were carried out for 5 min at 37°C, followed by two washes each with 2 mL of Krebs-Ringer-Henseleit buffer.

5. Cells were solubilized in 0.5 mL of 1% SDS, and radioactivity was determined using a Beckman Instruments (Palo Alto, CA) LS 6000 liquid scintillation counter at approx 50% efficiency. Cells from parallel wells were solubilized in 0.5 mL of 1 N NaOH for protein amount measurements using a Bio-Rad Protein Assay solution (Bio-Rad) (see Fig. 4 and Note 1).

0 0

Post a comment