Modulatory Role for Substance P: MATERIALS AND METHODS(3)

MATERIALS AND METHODS(3) Superfusion Experiments Tissues to be superfused were placed in a superfusion system similar to that developed by Porter and Licht. Posterior and intermediate lobes of the pituitary were discarded, and the anterior lobe was cut up in fragments of approximately 1-2 mm3. Similar numbers of these fragments were distributed in superfusion chambers filled with medium (effective volume 200 (ulI), which were then immersed in a water bath maintained at 37°C. Superfusion medium consisted of Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma) supplemented with 10 mM Hepes, 0.37% sodium bicarbonate, 0.1% BSA, 30 ng/L Tween 20 (Does-der, Barcelona, Spain), 1% penicillin-streptomycin solution (Sigma), and 0.01% bacitracin (Sigma). Chambers received culture medium at a flow rate of 5 ml/h controlled by a peristaltic pump. The effluent was led by output lines to a fraction collector. Preliminary studies were conducted in order to determine the time required to stabilize basal secretion of pLH. For this, 8 chambers were filled with pituitary fragments and superfused simultaneously for 48 h with DMEM. Medium corresponding to 1-h fractions was collected at 1, 2, 3, 4, 6, 8, 14, 18, 24, 36, 42, and 48 h, and the amount of pLH secreted in these fractions was quantified by EIA. After establishing the appropriate length for the preincubation period, experiments were carried out as follows: 8 chambers were superfused simultaneously; 2 of them received DMEM alone and were used as control; the other 6 chambers were separated into 3 groups of 2 chambers, each receiving, respectively, three 20-min pulses (separated by an intervening 100-min period) of DMEM containing 100 nM GnRH, 1 (xM SP, or a combination of both agents. Fractions were collected every 10 min and stored at -20°C until assayed for pLH content by EIA. buy ortho tri-cyclen
The mean number of cells per chamber at the end of superfusion, determined by using the dispersion protocol described above, was 0.96 ± 0.13 X 106 cells, with a cellular viability after superfusion of 76 ± 2.3%. Basal secretion was calculated for each chamber as the mean value of the 6 fractions collected before the time designated as 0 min, and the pLH response was expressed as pLH peak maximum reached after the pulse and as area under the curve (AUC). In order to avoid variability due to differences in the amount of tissue placed in each chamber, LH peak maximum was expressed as a percentage of the respective basal secretion. AUC was calculated by summing up the net values of pLH secretion, after subtracting the corresponding basal values. For each treatment, a total of 4 chambers were analyzed that were obtained in two separate, independent experiments carried out under identical conditions.

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