In P19 cells was investigated by measuring tetramethylrodamine methyl ester fluorescence

In P19 cells was investigated by measuring tetramethylrodamine methyl ester fluorescence by flow cytometry. P19 cells were seeded, differentiated and treated with melatonin at the concentrations described above. After, cells were trypsinized and loaded with 150 nM TMRM for 30 minutes and then evaluated for mean cell fluorescence by flow cytometry. The uncoupler carbonyl cyanide 4-phenylhydrazone was used as a control and was added 15 minutes after TMRM loading to cause mitochondrial depolarization. Oxygen consumption Oxygen consumption was measured polarographically with a Clark-type Oxygen electrode connected to a recorder in a thermostated water-jacketed closed chamber with magnetic stirring. The reactions were performed at 37C in 1 ml of glucose- or galacose-containing DMEM with 5 106 cells. Respiration was sustained with endogenous substrates followed by uncoupling by FCCP. Respiration rates were obtained assuming an oxygen concentration of 237 nmol O2/ml in the experimental medium at 37C. In order to confirm the measurement of mitochondrial basal respiration, 1.5 M rotenone was added to completely inhibit oxygen consumption by inhibiting mitochondrial complex I. Cell cycle analysis One million P19 cells were harvested, washed with PBS and fixed by adding 70% PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19861045 cold ethanol. Subsequently, cells were centrifuged at 850 g to remove the ethanol, washed twice with PBS and finally resuspended in 400 l PBS. 10 g/ml RNAse and 20 g/ml propidium iodide were added for 30 minutes at 37C. 20 103 events per sample were analyzed by using a FACScalibur flow cytometer with 488 nm excitation and 605 nm emission wavelengths. The percentage of cells in G1/G0, S and G2/M was determined using Modfit LT software. Adenine nucleotide measurement ATP, ADP and AMP levels were measured by HPLC. For adenine nucleotide extraction, after one rinse in cold PBS, 0.5 ml PBS and 0.5 ml perchloric acid/ EDTA were added to each dish. Cells were scraped from the dishes, placed in 1 ml micro-centrifuge tubes and centrifuged for 2 minutes at 14,000 g. Pellets were then re-suspended in 50 l of 1 M NaOH and later used for protein quantification by the BCA protein assay. The supernatant was neutralized with 3 M KOH in 1.5 M Tris and centrifuged again. The supernatant was again collected and stored at 80C until analyzed by reverse-phase high performance liquid chromatography. The chromatographic apparatus was a Beckman-System Gold, consisting of a 126 Binary Pump Model and a 166 Variable UV detector, controlled by computer. The Varlitinib detection wavelength was 17092 Oncotarget Intracellular calcium measurement Intracellular calcium levels were measured as described in the Fluo-4 Direct Calcium Assay kit. After seeding 5 105 P19 cells in 96 well plates, the medium was removed and 50 l of pre-warmed HBSS were added. The Fluo-4 Direct calcium reagent was prepared as suggested by the manufacturer and added in a www.impactjournals.com/oncotarget 254 nm, and the column was a Lichrospher 100RP-18 from Merck. An isocratic elution with 100 mM phosphate buffer, pH 6.5 and 1% methanol was performed with a flow rate of 1 ml/min. The time required for each analysis was 5 minutes. system and imaged with Versa Doc imaging system according to the manufacturers’ protocols. Densities of each band were calculated with Quantity One Software or ImageJ software. All data presented are representative from at least three separate experiments. Lipid peroxidation The MedChemExpress GW 501516 content in malondialdehyde was measured by.In P19 cells was investigated by measuring tetramethylrodamine methyl ester fluorescence by flow cytometry. P19 cells were seeded, differentiated and treated with melatonin at the concentrations described above. After, cells were trypsinized and loaded with 150 nM TMRM for 30 minutes and then evaluated for mean cell fluorescence by flow cytometry. The uncoupler carbonyl cyanide 4-phenylhydrazone was used as a control and was added 15 minutes after TMRM loading to cause mitochondrial depolarization. Oxygen consumption Oxygen consumption was measured polarographically with a Clark-type Oxygen electrode connected to a recorder in a thermostated water-jacketed closed chamber with magnetic stirring. The reactions were performed at 37C in 1 ml of glucose- or galacose-containing DMEM with 5 106 cells. Respiration was sustained with endogenous substrates followed by uncoupling by FCCP. Respiration rates were obtained assuming an oxygen concentration of 237 nmol O2/ml in the experimental medium at 37C. In order to confirm the measurement of mitochondrial basal respiration, 1.5 M rotenone was added to completely inhibit oxygen consumption by inhibiting mitochondrial complex I. Cell cycle analysis One million P19 cells were harvested, washed with PBS and fixed by adding 70% PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19861045 cold ethanol. Subsequently, cells were centrifuged at 850 g to remove the ethanol, washed twice with PBS and finally resuspended in 400 l PBS. 10 g/ml RNAse and 20 g/ml propidium iodide were added for 30 minutes at 37C. 20 103 events per sample were analyzed by using a FACScalibur flow cytometer with 488 nm excitation and 605 nm emission wavelengths. The percentage of cells in G1/G0, S and G2/M was determined using Modfit LT software. Adenine nucleotide measurement ATP, ADP and AMP levels were measured by HPLC. For adenine nucleotide extraction, after one rinse in cold PBS, 0.5 ml PBS and 0.5 ml perchloric acid/ EDTA were added to each dish. Cells were scraped from the dishes, placed in 1 ml micro-centrifuge tubes and centrifuged for 2 minutes at 14,000 g. Pellets were then re-suspended in 50 l of 1 M NaOH and later used for protein quantification by the BCA protein assay. The supernatant was neutralized with 3 M KOH in 1.5 M Tris and centrifuged again. The supernatant was again collected and stored at 80C until analyzed by reverse-phase high performance liquid chromatography. The chromatographic apparatus was a Beckman-System Gold, consisting of a 126 Binary Pump Model and a 166 Variable UV detector, controlled by computer. The detection wavelength was 17092 Oncotarget Intracellular calcium measurement Intracellular calcium levels were measured as described in the Fluo-4 Direct Calcium Assay kit. After seeding 5 105 P19 cells in 96 well plates, the medium was removed and 50 l of pre-warmed HBSS were added. The Fluo-4 Direct calcium reagent was prepared as suggested by the manufacturer and added in a www.impactjournals.com/oncotarget 254 nm, and the column was a Lichrospher 100RP-18 from Merck. An isocratic elution with 100 mM phosphate buffer, pH 6.5 and 1% methanol was performed with a flow rate of 1 ml/min. The time required for each analysis was 5 minutes. system and imaged with Versa Doc imaging system according to the manufacturers’ protocols. Densities of each band were calculated with Quantity One Software or ImageJ software. All data presented are representative from at least three separate experiments. Lipid peroxidation The content in malondialdehyde was measured by.