Lo que puede hacer la víctima en materia probatoria

The efficiency of transfection was checked by examining fluorescence images of transfected Sf9 cells expressing eGFP-RyR fusion constructs using a Leica M205 LA light microscope with GFP2 filter (Leica, Wetzlar, Germany), and similarly transfected HEK 293 cells with a Zeiss Axio Vert fluorescence microscope x10 objective (Zeiss, Oberkochen, Germany). This step was omitted for untagged RyR constructs. High magnification images were taken using either a Leica SP5 x63 oil immersion lens or Zeiss 780LSM x63 and x40 water immersion lenses. HEK 293 and Sf9 cells were imaged both in vivo in culture dishes and fixed onto coverslip with 0.4% formaldehyde.

4.2.4.1 Calcium release assays for HEK 293 cells

Expression of full-length P. xylostella and M. persicae constructs, and the P. xylostella deletion mutant in HEK 293 cells, was determined by monitoring the release of calcium from internal stores with a calcium sensitive fluorescent dye after addition of RyR agonists. 1x105 HEK 293 cells were seeded onto glass bottom dishes (MatTek, MA, USA) (12mm diameter) and transfected with plasmid DNA containing the RyR cDNA using Effectene® as follows (protocol to prepare 7 dishes): plasmid DNA (0.8µg) was diluted with 6.4µl of enhancer in 100µl of EC buffer (both supplied with Effectene®) and the mixture was incubated for 5 minutes at room temperature. After 5 minutes, 20µl of Effectene® was added and the mixture incubated for a further 10 minutes. In the meantime cells were washed with complete DMEM medium (cDMEM, containing 10% FBS and penicillin/streptomycin 100U/100µg/ml). Then 600µl of cDMEM was added to the plasmid DNA / Effectene® mixture and thoroughly mixed by vortexing. 100µl of this solution was then applied to each dish and the cells were returned to the 37oC / 5% CO2 incubator.

HEK 293 cells expressing human RyR2 were used as a positive control for the calcium imaging experiments and Fluo-3 AM (Life technologies, CA, USA) loaded un-transfected HEK 293 cells were used to test for potential excitation of endogenous calcium stores. Cells in 200µl DMEM were loaded with 2µl of a 1mM

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solution (final conc. 10µM) of Fluo-3 AM calcium sensitive dye, 24 and 48 hours post transfection, and incubated for 45 minutes at 30oC / 5% CO2 to avoid dye

compartmentalization. After the loading was completed, cells were incubated for 10 minutes with 2ml of fresh DMEM media. Calcium release events were imaged using an SP5 confocal microscope (Leica, Wetzlar, Germany) with the cells in 200µl of fresh DMEM. 60 second recordings were made, one recording per dish per agonist, measuring excitation at 488nm and capturing emissions at 525 nm every 70ms.

4.2.4.2 Calcium release assays in Sf9 cells

Fura 2-AM dye (Life technologies, CA, USA) was used for monitoring calcium release in Sf9 cells transfected with the untagged P. xylostella RyR. Glass coverslips (1cm diameter glass) coated with Poly-L-lysine (Sigma, MA, USA) were placed in a 4 well plate. Each well was then filled with 500µl of Sf-900 II medium and each coverslip was seeded with 2.5x105 cells. Cells were allowed to attach to the coverslips for 1h and then they were transfected in 4 well plates using the optimised Cellfectin™ II method (discussed later in section 4.3.2.3). 24h, 48h, 72h and 96h post transfection cells were loaded with 1mM of Fura 2-AM. Cells on coverslips in 4 well plates were first put into 500µl of fresh SF-900 II medium and then 2µl of the dye stock solution (5mM) was added. Cells were left to incubate at 27oC for 45-60 minutes, followed by 3 washes with 500µl of fresh un-supplemented Sf-900 II medium. Prior to imaging, coverslips with Fura 2 loaded cells were placed in standard Ringer’s solution (recipe in Appendix A, section 8). All imaging was done using an Axio Vert.A1 microscope with a LD Plan-Neo Fluar x20/0.4 lens (Zeiss, Oberkochen, Germany), measuring the ratio of excitation at 340/380nm (calcium free/calcium bound indicator) every 180ms and capturing emission at 510nm for at least 60 seconds. Cells on the coverslip were placed into a perfusion chamber of approximately 0.5 ml volume mounted on the microscope stage which was connected to a peristaltic pump, allowing for a constant fluid exchange (flow rate 3 ml/min). Test solutions were applied using 3-5 seconds bursts via a glass u-tube (Figure 20).

Chapter IV

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Figure 20 Perfusion chamber setup with a glass U-tube applicator for recording of multiple agonist applications. Black arrows indicate flow of Ringer’s solution, which allows for a constant replacement of solution in the perfusion chamber. Agonist solutions are applied via the glass U-tube, where flow is controlled by recording software (VisiView®) or the electrical switch on the the peristaltic pump. When the U-tube flow is stopped, agonist present inside the tube diffuses over the cells by seepage through the application hole.

Experiments were recorded using VisiView® software (Visitron Systems, Puchheim, Germany) and the numerical data analysed using Microsoft Excel 2010 and SigmaPlot v.12 (Systat Software). The data were normalized using the equation: R/R0, where R is a fluorescence ratio value recorded for individual time points and

R0 is an average fluorescence ratio calculated over the first 5 seconds prior to

addition of the agonist. Data points on graphs are presented as the mean values of individual cell responses for each independent experiment and standard errors compared to the mean. The normalized amplitude responses of individual cells were calculated by estimating the highest value subtracted from the basal level (Rmax–

R0=∆Rmax) and normalizing with the equation: ∆Rmax/R0. Final amplitude data was

presented as a mean value of all cells in the individual experiment and the standard deviation of the mean.

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