Cultures had been maintained 48 h before working with them in experiments, to permit good recovery after the isolation method. With this particular strategy we normally obtained 1 106 cells per mouse, purity was assayed by RT PCR and immunocytochemistry against CYP11A protein, To exclude contamination with granulosa, the expression in the FSH receptor transcript as well as responsiveness of CREB phosphorylation to hCG or FSH had been assayed. Reverse Transcription Polymerase Chain Response Total RNA of TIC cultures or through the indicated organ was purified using the guanidine isothiocyanate system, Initially strand cDNA was synthesized employing two ug of DNase taken care of RNA as template, 1 mg of oligo, ran dom hexamers, and reverse transcriptase. The cDNA was used as template in a polymerase chain reaction to amplify cDNA fragments for B actin, p2y2r, p2y4r, and p2y6r transcripts, and for cyp11A, cyp17A, star, and fshr as good and damaging theca cell markers, respectively.
The many PCR packages began at 96 C for 3 min and finished at 72 C for one min. The amplification cycles consisted in forty s at 96 C, 40 s in the specific annealing temperature for each primer set, and forty s at 72 C. The amplified merchandise had been gel isolated, phenol chlo roform purified, and subcloned into the pCR4 TOPO vector, Their nucle otide Chk1 inhibitor sequences were confirmed by automatic sequenc ing. Fluorescence microscopy Mouse ovarian TIC were grown on 12 mm diameter cover slides. Semi confluent cultures have been loaded for 15 min with five mM fluo four AM and 0. 1% pluronic acid in Krebs remedy, The cells have been washed with Krebs resolution for ten min to elim inate excess dye and then positioned inside a frequent movement recording chamber that allowed them to be visualized with an inverted fluorescence microscope, Medicines were applied by superfusion and responses have been recorded with an Evolution QEi cam era, Sequences of images had been analyzed using the Image Professional Plus application and Imagenes soft ware, a program produced exclusively for this analysis, From the Ca2 free Krebs option, CaCl2 was replaced by three mM MgCl2.
Western blot For MAPK p42 and p44 or CREB phosphorylation exper iments, cultured TIC were harvested 24 h before the experiment to reduce serum dependent kinase activ NU7441 ity. Just after that, cells were stimulated with the indicated medicines, scraped in Laemmli buffer, and boiled for 5 min. For electrophoresis, samples had been fractionated within a 10% SDS polyacrylamide gel and transferred to a nitrocellu get rid of membrane, Membranes have been blocked for one h at room temperature in 150 mM NaCl, twenty mM Tris, pH seven. four, and 0. 1% Tween twenty containing 5% nonfat dry milk then incubated more than evening at 4 C using the ideal rabbit principal antibody directed towards the phosphorylated form of MAPK p44 and p42 or CREB, Right after washing with TBS T, membranes had been incu bated 1 h at 37 C with HRP conjugated goat anti rabbit antibody in TBS T.