ESTABLECIMIENTO DE CONDICIONES ADECUADAS PARA LA OBTENCIÓN Y CARACTERIZACIÓN DE

2.8.1 The preparation of RBL-2H3 cells for the measurement of |Ca^^];

Free cells were prepared from a RBL-2H3 monolayer cell culture. They were firstly washed with PBS and then harvested by trypsinisation ( See section 2.3.1). The cell suspension in trypsin-EDTA solution was centrifuged at 225g for 3min and then resuspended in fresh media. Cell counting indicated a harvested cell density o f ~ 5x10^ cell/ml. 0.5ml of this cell suspension was then placed within the ‘calcium recording chamber’ which consisted of a glass ring (diameter 16mm; height 3mm) attached to a microscope slide coverslip using a silicon rubber elastomer ‘Sylgard’. During the culturing procedure the recording chamber was kept in a standard 35mm culture dish within a humidified incubator (5% CO] and 95% air) at 37°C. Cells were nomially used within 48 hours after culture. The cells were sensitised to antigenic stimuli by incubating overnight at 37°C with the DNP specific monoclonal mouse IgE antibody (0.5pg/ml).

2.8.2 Indo-1 loading

Cells were loaded with the Ca indicator dye, indo-1, by using the acetoxymethyl ester form; indo-1 AM [231]. Cells were incubated with IpM indol-A M for 30-45 min in the culture medium at 37°C. To assure adequate dispersion of both indo-1 and its DMSO solvent, lOOpl of culture media was removed from the recording chamber and transferred into a 0.5ml Eppendorf tube where 0.5pl of indo-1 AM (Im M in DMSO) was added. This was then mixed using a vortex and returned into the recording chamber. The uncharged lipophylic indo-1 AM readily passes into the cell cytoplasm where endogenous esterases induce ester hydrolysis generating indo- 1 free acid, which being charged is trapped within

the cell. Indo-1 had no effect on the spontaneous and antigen-stimulated 8-

hexosaminidase release from RBL-2H3 cells (data shown in section 6.3.1).

2.8.3 The recording of [Ca^^Ji from RBL-2H3 ceils

The method used in this study was as previously described by Marsh et al. (232, 233, 234, 235]. The recording chamber was placed onto the microscope stage of a Nikon inverted microscope (Diaphot) fitted with UV epifluorescence illumination.. The recording chamber was continuously perfused with the modified Tyrode’s buffer (8-12 ml/min at 37°C ). To record [Ca^^]j, following indo-1 loading (see above), individual cells were excited with UV light (360nm) and emitted light at both 408 and 480 nm was recorded simultaneously using a series of dichroic mirrors and photomultiplier tubes

(Thorn EMI QL30) (see Fig. 2.5) A home-built amplifier converted the anodal

photomultiplier current to voltage and continuously output the ratio of the emissions at 408 and 480nm (408/480 ratio). The 408/480 ratio output was computer digitised at 2.5 Hz using a Digidata 1200 and ‘pClamp 6.0’ software (Axon Instruments ) and the estimation of [Ca^^], undertaken on-line after subjecting the data to a simplified algorithm (see below).

2.8.4 Calculation of [Ca^^]i and calibration of the system

[Ca^^li was estimated from the acquired ratio data using the equation first described by Grynkiewicz at at. in 1985 [231] :

[Ca

ji = [(R - Rmin )/(Rmax - R)| x K^(F„/Fs)

Where, Kd is the dissociation constant for Ca“^ and indo-1, F,/Fs is the maximum excursion at 450nm,

R is the recorded 408/480 emission ratio, Rmin is the minimal value of R at zero [Ca^^]„ and Rmax is the value of R at saturating [Ca^^]i. Rmin=0.38, Rmax= 4, Kd(F,/F, )=1400nM

The measured constants used in the above equation were obtained in a separate set of experiments carried out by Dr. Steve Marsh (Pharmacology U.C.L) by applying a series of known intracellular calcium concentrations in the presence o f indo-1 to the

inside of sympathetic neurons using a whole cell patch pipette and then measuring the 408/480 ratio. This allowed construction of a calibration curve and estimation of the above constants. H.V. SOURCE F 2 .-4 8 0 n m DiC-430nm 480 AMP IN D O -1 P M T 1. EXCITATION F2-408 nM 360nm ^ N O N DiC-400nm EMISSION RATIO AMP 408 AMP MIRROR COMPUTER Figure 2.5: Schematic diagram of [Ca^^Jj measuring equipment.

2.8.5 Quantification of the data

In this study antigen-induced changes in [Ca^^]j are oscillatory, asynchronous and irregular in individual RBL-2H3 cells. Figure 2.6 shows Ca^^ oscillations induced by 1 ng/ml DNP-HSA in 18 different cells. The so-called Ca^^-fingerprint is obvious and the antigen-induced changed in [Ca^'^Ji in individual cells is different in frequency, amplitude and shape (Fig. 2.6). Millard et al. have used a graph obtained from averaging the response of more than ten cells to quantify their data [114]. Their method is not suitable for this study as it does not allow the evaluation of the effect of drugs on the antigen- stimulated changes in [Ca^^]i. However in this study, to address this problem, the area under the curves (AUC) have been calculated, using the ‘Origin 4 ’ software program. To enable a comparison between the control and the effect of drugs and the estimation of the antigen-induced changes in [Ca^^]j, I utilised an integration procedure on the [Ca^'^jj response using the ‘Origin 4 ’ software program to generate the area under the curve (AUC). Before calculation of AUC, the resting [Ca^^Ji on the curves was adjusted to zero, and data was presented solely for the increased [Ca^^Jj. The equation used for calculating AUC was:

i = 1535 2 ( Y j + Y i + 1 )

A X

In detail, an oscillation curve was divided into 1536 sections, the area for each section calculated as a trapezoid whose heights are Y, and Yj+i and its base equal to the interval time between each two samples (Ax=0.4second). Finally the calculated area for all sections were summed (see Fig. 2.7). The above equation is a general formula for this calculation method.

Data obtained from the ‘Origin 4 ’ software program or calculated from the above equation, using ‘Microsoft Excel’, were exactly identical.