Transfection of CHO-hY2-K9 cells

CHO-hY2-K9 cells were seeded in 500 µl of Ham’s F12 supplemented with 10 % FCS on a 24-well plate. On the day of transfection, 60-70 % confluence was reached. The pcDNA3.1/Hygro-qi5 vector was linearized with Eam11051 for 1 h at 37 °C and subsequently purified with the Quiagen PCR purification kit (Quiagen). The DNA was assumed to be 90 % of the amount of the applied DNA as the manufacturer specifies 90-95 % recovery of the DNA after purification. For transfection, 300 ng of linearized plasmid and 1.8 µl of FuGENE™ were used per well. Transfections were performed according to the manufacturer’s instructions. Medium was replaced 24 h after transfection. Two days post-transfection, the cells were trypsinized and transferred into 25-cm² tissue culture flasks (Becton Dickinson, Franklin Lakes, N.J., USA) with Ham’s F12 containing 10 % FCS, 400 µg/ml of geneticin and 400 µg/ml of hygromycin, respectively. Untransfected CHO-hY2-K9 cells were maintained in the same selective medium as a negative control. Cells were passaged by 1:10 splitting every 3 days. After 3 weeks control cells were dead and single clones of the transfected cells were selected as described in 3.1.2.12 and expanded for analysis in a flow cytometric calcium assay.

4.1.2.4 Flow cytometric calcium assay

Cells were grown for 2 days to 70-90 % confluence, trypsinized and detached with Ham’s F12 supplemented with 10 % FCS to inactivate trypsine. Cells were counted in a hemocytometer, centrifuged for 5 min at 300 g at room temperature and resuspended at a density of 2.66 ·10⁶ cells / ml in loading buffer (Gessele, 1998) containing 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1.5 mM CaCl2, 25 mM HEPES and 10 mM glucose at pH 7.4. For the preparation of the loading suspension, 3 µl of

fluo-4-AM (Molecular Probes; 1 mM stock solution in anhydrous DMSO) were added to 5 µl of pluronic™ F-127 (Molecular Probes; 20 % stock solution in DMSO) and mixed carefully before addition of 1 ml of loading buffer containing 2 % BSA. 330 µl of loading suspension were added to 1 ml of cell suspension resulting in a cell number of 2 · 10⁶ cells / ml and a dye concentration of 0.7 µM.

The cells were incubated in the dark for 30 min at room temperature and recentri-fuged at 300 g for 5 min. After resuspension in loading buffer at a density of 0.5 - 1 · 10⁶ cells/ml, the cells were incubated again for 30 min at room temperature in the dark; during this postincubation step, the AM-ester is intracellularly cleaved and thus the calcium indicator is trapped in the cell.

O O N

O

O H₃C O

O O O H₃C O

(CH₂)₂ O

CH₃ N O O

O H₃C

O

O O

O H₃C

O

O O

F F

O O O H₃C

Measurements were performed in a purpose-built glass tube closed by a silicon septum as described (Schneider, 2005). This instrumentation allows injections into the samples during continuous flow cytometric measurements. A tube containing 1 ml of the cell suspension was connected with the flow cytometer under permanent stirring and the recording was started. Instrument settings were: FSC: E-1; SSC: 280;

FL-1: 350; flow: high.

After 30 s of measurement of the basal fluorescence 10 µl of peptide agonist solution were injected with a hamilton syringe and data were recorded for another 90 s. The needle of the flow cytometer was washed with millipore water after each measurement. Raw data were first averaged with the WinMDI software and then exported to Sigma Plot™ 8.0.

Fig. 36: Structure of the fluo-4 AM ester.

Data were further smoothed (running average) with SigmaPlot™. The level of increase in fluorescence was calculated from the difference between the baseline (mean fluorescence of the first 25 s) and the highest value of the averaged curve.

These amplitudes of the averaged signals were used to construct concentration-response curves. For the determination of EC50 values of agonists, every third measurement was a 100 % reference signal elicited with 1 µM pNPY.

Dilutions of Y2 receptor antagonists (Fig. 18) were made in DMSO, and 10 µl of antagonist was preincubated with 990 µl of cell suspension for 1 min before the measurement. Calcium response was triggered with 10 µl of 70 µM pNPY in 10 mM HCl containing 0.1 % BSA. The 100 % reference signal was induced in every third measurement, too. In this case, cells were preincubated in the presence of the solvent without antagonist. EC50 and IC50 values were calculated with Sigma Plot™

(Version 8.0, SPSS Inc.) using the equation of the four parameter logistics function.

Fig. 37: Flow cytometric calcium assay. Fluorescence of fluo-4-loaded cells was recorded in channel 1 (FL-1) over 2 min. After 30 s, injection of 100 nM pNPY causes an increase in fluo-rescence of gated qi5-trans-fected CHO-hY2-K9 cells.

Values were averaged with WinMDI software (blue line).

time [s]

0 20 40 60 80 100 120

fluorescence in FL-1 [RFU]

1 10 100 1000 10000

4.1.2.5 Spectrofluorimetric calcium assay

The spectrofluorimetric calcium assay with the ratiometric Ca²⁺ indicator fura-2 was performed as described for HEL cells by Gessele (Gessele, 1998). Cells were grown to 70-80 % confluence, trypsinized and resuspended in FCS containing medium for trypsine inactivation. Cells were counted, centrifuged at 300 g for 5 min and resuspended at 1.3 · 10⁶ cells/ml in loading buffer. 0.75 ml cell suspension were added to 0.25 ml of loading suspension containing 20 mg of BSA, 5 µl of pluronic F-127 (20 % in DMSO) and 4 µl of fura-2/AM (Molecular Probes; 1 mM in anhydrous DMSO) in 1 ml of loading buffer. The addition of pluronic F-127 facilitates the solubilization of the lipophilic calcium indicator dye and the following dye loading as described for fluo-3 in (M. E. Granados, 1997) and (Kao et al., 1989). Final concentrations were: 1· 10⁶ cell/ml, 1 µM fura-2/AM, 0.2 % DMSO and 0.025 % pluronic F-127. The cells were incubated for 30 min at room temperature in the dark, centrifuged and resuspended in the same volume of loading buffer. In order to achieve complete intracellular cleavage of the AM-ester, the cells were incubated for additional 30 min in the dark, washed twice with loading buffer and resuspended at a density of 1 · 10⁶ cells/ml.

For the measurement, 1 ml of the cell suspension was transferred into a cuvette containing 1 ml of loading buffer under stirring. The baseline was recorded for 30 s before the agonist was added. Antagonists were added to the cell suspension 1 min before the calcium signal was triggered by the addition of a fixed concentration of

Fig. 38: Structure of the fura-2 AM ester.

agonist. Every third measurement was a reference signal. For the determination of agonist activity, the reference signal was triggered by 1 µM pNPY; for the determination of IC50 values of antagonists, the reference signal was elicited in the absence of antagonist. Instruments settings were λex = 340 and 380 nm (alternating) with slit = 10 nm and λem = 510 nm with slit = 10 nm. Stirring was low and temperature was 25 °C.

The ratio R of fluorescence intensity at 510 nm after excitation at 340 and 380 nm was used for the calculation of the calcium concentration according to the Grynkiewicz equation (Grynkiewicz et al., 1985):

The KD value is the dissociation constant of the fura-2-Ca²⁺ complex. Rmax is the fluorescence ratio in presence of a saturating Ca²⁺ concentration, determined after the addition of 10 µl of digitonin solution (2% in water, Sigma), which caused lysis of the cells and saturation of the dye with the calcium ions of the loading buffer. Rmin is the ratio in absence of free Ca²⁺, determined after the addition of 50 µl of EGTA solution (600 mM in 1 M tris buffer, pH 8.7) to the lysed cells. The correction factor SFB is the ratio of fluorescence intensity at 510 nm after excitation at 380 nm of the Ca²⁺ free and Ca²⁺ saturated dye.

[ ]

R) (R

) R K (R

Ca

max min D

2 ⋅

−

⋅ −

+ =