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2.5.1. Assay for P produced by isolated cytotrophoblast cell incubates

Triplicate tubes were prepared for a standard curve containing 0, 31.8, 63.6, 127.2, 254.4, 508.7, and 1017.5 pmol/1 P and dried down at 40°C using a stream of air produced by a pump.

Appropriate volumes were taken from the culture wells and pipetted into glass extraction tubes with the addition of a suitable volume of carbonate buffer (1:1 carbonate buffer to sample) and diethyl ether was then added to the tubes such that the ratio of diethyl ether to sample was at least 20:1. A series of tubes was prepared which contained only the carbonate buffer solution and diethyl ether. All tubes were vortexed for 10 minutes and frozen at -20°C for 20 minutes. The supernatant was then carefully decanted into an appropriately labelled test tube and dried down at 40^C with an air pump. The supernatants of those tubes containing only carbonate buffer and diethyl ether were decanted into blank tubes. Five hundred |xl assay buffer was added to all tubes before vortexing for 5 minutes. Appropriate aliquots were taken from each sample tube. The same volume was transferred from the blank tubes to those containing the standards. The volumes in all the tubes were then standardized to 400 jj.1 with assay buffer. The tubes containing the standards were then vortexed for 5 minutes and the radioactivity and antiserum were added to all the tubes as described previously in section 2.4.1.

2.5.2. Assay for E l produced by isolated cytotrophoblast cell incubates

Duplicate volumes were taken from the wells and transferred to labelled glass extraction tubes and diethyl ether was added in a ratio of at least 20:1 (diethyl ether to sample). A number of tubes containing just diethyl ether were also included. All tubes were vortexed for 10 minutes and frozen at -20°C for 20 minutes. The supernatant was then decanted into labelled glass reaction tubes. Those tubes containing diethyl ether alone were decanted into tubes containing the E l standards, in triplicate, at concentrations of 18.5, 37.0, 74.0, 147.9, 295.9, 591.7, 1183.4 pmol/1. After drying down at 40°C 400 |il buffer was added and the tubes were vortexed for 5 minutes. The assay procedure was then carried out as previously described in section 2.4.2.

2.5.3. Assay for £ 2 produced by isolated cytotrophoblast cell incubates

Duplicate volumes of the samples were pipetted into labelled extraction tubes and carbonate buffer was added in a ratio of 2:1 (buffer to sample). Diethyl ether was added in a ratio of at least 20:1 (diethyl ether to sample). The tubes were vortexed for 10 minutes, frozen at -20°C for 20 minutes and then the supernatants were decanted into the respective tubes. The tubes containing only carbonate buffer and diethyl ether were decanted into the tubes containing the E2 standards, in triplicate, at concentrations of 0, 18.2, 36.4, 72.9, 145.8, 291.6, 583.1 and 1166.2 pmol/1. All the tubes were dried down and 400 pi assay buffer added. All the tubes were vortexed for 5 minutes and the assay procedure was identical to that described for the tissue incubates in section 2.4.3.

2.5.4. Assay for E3 produced by isolated cytotrophoblast cell incubates

Appropriate volumes of samples were pipetted into labelled glass extraction tubes and carbonate buffer was added in a ratio of at least 2:1 (buffer to sample). Certain tubes to be decanted into the tubes containing the standards contained only carbonate buffer. Diethyl ether was added in ratio of at least 20:1 (diethyl ether to sample) and the tubes were vortexed for 10 minutes before being frozen at -20°C for 20 minutes. The supernatants were decanted into the respective tubes. The tubes containing only carbonate buffer and diethyl ether were decanted into the tubes containing E3 standards, in triplicate, at concentrations of 0, 17.3, 34.7, 69.4, 138.7, 277.4, 554.8 and 1109.6 pmol/1. All the tubes were dried down and 400 pi assay buffer added. The supernatants were decanted, dried down and 400 pi assay buffer was added. All the tubes were vortexed for 5 minutes and the assay procedure was then identical to that described for the tissue incubates in section 2.4.4.

2.5.5. Assay for HCG produced by isolated cytotrophoblast cells

The assay was performed in accordance with the information provided with the kit and all reagents were made up accordingly. All standards, controls and samples were assayed in duplicate.

The standard curve (concentrations 0 ,5 , 10, 25, 100 and 300 mlU/ml) was prepared by pipetting 50 pi of the standards provided to the bottom of polypropylene tubes. Two controls provided with the kit were included in each assay. Appropriate aliquots were taken from the medium in which the cells had been incubated and pipetted into labelled polypropylene tubes. The volume was then made up to 50 pi with DMEM. Iodinated antiboby solution (200 pi) was added to each standard, sample and control. The tubes were vortexed and an avidin coated bead was added to each. The tubes were then incubated at room temperature for one hour. During the incubatory period the tubes were shaken by hand 100 times every 15 minutes. After incubation the beads were washed twice in a solution provided with the kit and the liquid aspirated from each tube. The tubes were counted on a gamma counter for one minute and the amount of HCG was computed by logit/log transformation.

2.5.6. Assay for HPL produced by isolated cytotrophoblast cells

The assay was performed in accordance with the information provided with the kit and all reagents were made up accordingly. All standards and samples were assayed in duplicate.

A standard curve was prepared by pipetting 50 pi of the standards provided (concentrations 0.0, 0.025, 0.1, 0.25, 0.5 and 1.0 pg/ml) to the bottom of duplicate polypropylene tubes. Aliquots (50 pi) of the medium in which the cells had been incubated were pipetted into appropriately labelled tubes. Iodinated HPL (1 ml) was

added to each standard and sample. The tubes were vortexed and incubated at 37°C for three hours. The liquid from each tube was decanted simultaneously. Each tube was counted on a gamma counter for one minute and the amount of HPL was computed by logit/log transformation.

2.5.7. Assay for ACTH produced by isolated cytotrophoblast cells

The assay was performed in accordance with the information provided with the kit and all reagents were made up accordingly. All standards, samples and controls were assayed in duplicate.

A standard curve was prepared by pipetting (200 )il) of the standards provided (concentrations 0, 5, 14, 50, 140, 465 and 1400 pg/ml) to the bottom of labelled polypropylene tubes. Two controls provided with the kit were also pipetted in the same manner. Aliquots (200 pi) were taken from the medium in which the cells had been incubated and pipetted into labelled polypropylene tubes. Iodinated ACTH antiboby solution (100 pi) was added to each standard, sample and control. The tubes were vortexed and an avidin coated bead was added to each. The tubes were then incubated at room temperature for twenty hours. After incubation the beads were washed twice in a solution provided with the kit and the liquid aspirated from each tube. The tubes were counted on a gamma counter for one minute and the amount of ACTH was computed by logit/log transformation.