SENSIBILIDAD AL AGUA DEL COPOLÍMERO DE VINIL ACETATO

was used, as it poses as a good substitute for in its passage through

channels and has the advantage over radioactive (e.g. of having a much longer

half-life (18 days). This enables a sufficient length of study before the p-emission decays beyond the countable range (normally 3-4 half-lives).

2.7.1 Cell preparation

Passively sensitised RBL-2H3 cells were prepared as described in section 2.6.1, but at the end, the cell pellet was resuspended in fresh medium with a count of 4x10^ cell/ml.

2.7.2 uptake measurement experiment

To evaluate the and to study the time course of ^^Rb^ loading, 7ml of cell suspension containing 4x10^ cell/ml were loaded with ^^Rb"^ (3pCi/ml)^ at 37°C in a shaking incubator for 3 hours. During the incubation time, 0.5ml samples were taken at 15 min periods (as ^^Rb"^ loading is a relatively slow process) from ^^Rb^ addition. The samples were centrifuged at 225g for 3min and the pellets were resuspended in 5ml perchloric acid (0.4M) and transferred into scintillation vials. The ^^Rb"^ loaded into cells was counted in a liquid scintillation counter. Figure 2.2 shows the time course of ^^Rb^ loading in RBL cells. 50% total uptake occurred at 30 min and reaches a plateau at approximately two hours.

2.7.3 loading

Cells in susp

shaking incubator for 2 hours. As already noted, uptake has a doubling time of 30min and reaches a plateau at approximal

statistically significant (P<0.1).

Cells in suspension (4x10^ cell/ml) were loaded with ^^Rb^(3pCi/ml) at 37°C in a

reaches a plateau at approximately 2 hours. Increases in loaded ^^Rb"^ after 120min are not

M ethods & Materials 35000-1 30000 - 25000 - 20000 -

i

Figure2.2: Time course study of loading into RBL-2H3 cells. Cells (4x10^ cell/ml) were incubated with 3pCi/ml ^Rb^ at 37°C. samples were taken at 15 min intervals and spun down. The pellet was resuspended in perchloric acid (0.4 M) to lyse the cells. The “loaded '^Rb ” was counted on a liquid scintillation counter. CPM changes after 120min are not statistically significant (P<0.1).

2.7.4 efflux measurement experiment

For a typical experiment, 0.5ml of cell suspension (4x1 O'" cell/ml) loaded with

^Rb^(see section 2.7.3) was centrifuged (at 225g for 3min), resuspended in 2ml modified Tyrode’s (0.05% BSA) and preincubated (1x10^ cell/ml) for 1 min at 37°C in a shaking water bath prior to the start of each experiment. Prior to initiation of the experiment, test drugs were pipetted into the test tubes at 1 0 0 times the test concentration and then the

cell suspension was added into the test tube to dilute the test drug to the desired concentration. Cells were then incubated with drugs for 9 min before antigen stimulation. Each experiment was carried out for a total of 24 minutes, stimulant (antigen or A23187) being added after 8 minutes in order that a basal rate of ^'"Rb^-efflux be established.

Samples were taken every 2 min from 8 min before (to establish the spontaneous rate of

^^’Rb^-efflux), to 16 min after, antigen stimulation'^ (to calculate the stimulated rate ^^Rb^- efflux). lOOpl aliquots of the cell suspension were taken at each time point and sedimented (at 16000g for lOsec) through 200pl phthalate oil (60% dibutyl phthalate and

Secretion w as measured for 15min and 16min for this experiment gives a desired comparison 2min time intervals have chosen for logistical reasons.

40% phthalic acid bis(2-ethylhexyl ester) using an Eppendorf microfuge. 50pl of the supernatant on top of the oil was transferred into scintillation vials containing 5ml distilled water. In order to lyse the cells and obtain a standard for total ^^Rb"^ uptake for each experiment, 5 ml perchloric acid 0.4M was added to a tube containing the same cell number after centrifuging and discarding the media. The ^^Rb^ released by cells was counted on a liquid scintillation counter.

2.7.5 Quantification of the data

Since a plot against time of the total amount of ^^Rb"^ lost produces a curve, it was preferred to use a linearising equation which would allow an estimate of the rate constant to be obtained. A logarithmic function was used:

In (Q.- A Q/Q.)

k= rate of ^^Rb^-efflux t= time,

Q.= total ^^Rb"^ uptake by the same number of cells during the loading time, which is the cpm recorded from the perchloric acid-treated cells.

A Q= the amount of ^^Rb^ released from cells into the supernatant which equals the cpm

recorded from each sample at any time point.

Now, if -ln(Q.- A Q/Q.) is plotted against time, a line can be obtained whose slope

is k (Fig.2.3). The other advantage of this equation is to normalise results from different experiments, irrespective of variation in the number of cells or ^^Rb^ loaded into the cells.

For each experiment the data from T= - 8 min (8min before addition of stimulant)

to T=0 (just before stimulant addition) were plotted and the slope of the regression line for this period gave an estimate of the rate constant for spontaneous ^^Rb^ efflux. When test drug has been used, the calculated rate from T= - 8 to T=0 was compared with control,

to show the effect of drugs on the spontané analyse these data was “Microcal Origin 4” .

to show the effect of drugs on the spontaneous ^^Rb^ efflux. The best software found to

To express the effect of test drugs on the stimulated ^^Rb^-efflux, two different methods have been used.

1.To express as a value, the initial rate of stimulated ^^Rb^-efflux which was obtained from the difference between the rate of ^^Rb^-efflux after cell stimulation and the rate of spontaneous ^^Rb^-efflux. The calculated initial rate constant o f stimulated ^ W -e fflu x for lOng/ml DNP-HSA was 0.015298 ± 0.001291 m in T h e r e f o r e , the inhibitory effect of tested drugs were calculated by comparison of the stimulated rate of ^^Rb^-efflux with and without drugs.

2.To present the data graphically, data points from a linear regression fit were

subtracted from the corresponding values for the efflux after stimulation (from t= 0 to

t=16) (Fig. 2.4). The advantage of this curve, in addition to showing the effect o f drugs, is that it also gives an indication of the time course o f stimulated ^^Rb^ efflux from the ‘control’ cells. DNP-HSA [l()ng/ml| Stimulated efflux Rate = 0,0297 min ■ DNP-HSA llOng/mi] 0.8 -

Sponta neous *^Rb+ efflux Rate = 0.0156 min i

0 .7 -

0 .6 -

Tim e (m m )

Figure 2.3: Antigen caused increase in ’^Rb^-efflux in sensitised RBL-2H3 cells. Cells were preincubated in tnodified Tyrode’s buffer 9 min before stimulation at 37°C. Samples were taken after one min from Smin before to 16 min after antigen addition. Antigen [DNP-HSA lOng/ml] was added at 0 min. The red line represent the spontaneous ^^Rb-efOux data points.

M ethods & Materials 0 . 3 0 -n * 0 25 - E_o> 0^ 0 . 2 0 1 0. 15 - •a_<u flj " 5 01 0- S ^ 0. 0 5 H 0.00 - DNP-HSA [lOng/mlJ^ -4 0 4 T im e ( mi n) 12 16

Figure 2.4: ^Rb-elTlux stimulation by antigen (DNP-HSA lOng/ml] in sensitised RBL cells. Stimulated '^Rb efflux at each point is defined as the difference between the value for the efflux after triggering (from t=0 to t=16) and the values calculated from a linear regression fit of basal efflux (the first five timepoints from t=-8 to t=0 on the red line in Fig.2.3).