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  • br Experimental design materials and methods

    2018-11-07


    Experimental design, materials and methods
    Material availability and repository The expression vectors described in this work are part of the SEVA initiative (http://wwwuser.cnb.csic.es/~seva/, [2,17]) and are available free of charge upon request.
    Acknowledgments Authors are indebted to Svein Valla (Norwegian University of Science and Technology, Trondheim, Norway) for his tips on the use of the ChnR/P expression system. This work was supported by the CAMBIOS Project of the Spanish Ministry of Economy and Competitiveness (RTC-2014-1777-3), the ST-FLOW (FP7-KBBE-2011-5-289326), EVOPROG (FP7-ICT-610730), ARISYS (ERC-2012-ADG-322797), and EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536) Contracts of the European Union, and the PROMPT Project of the Autonomous Community of Madrid (CAM-S2010/BMD-2414). The authors declare that there are no conflicts of interest.
    Data The first experiment to probe proton caged compounds (PCCs) as tools to manipulate and monitor the intracellular pH was performed by dosing the 1-(2-nitrophenyl)-ethylhexadecyl sulfonate (HDNS) to 3T3-NIH trpv1 antagonist [1] and observing the effects on a single cell. PCCs yield one proton per molecule, therefore the intracellular proton release is related to the amount of PCCs that can be conveyed into the cells. An enhanced uptake can be obtained by vectorization of PCCs with gold nanoparticles (AuNPs) [2], or intervening on the cellular permeability, for instance, with DMSO. We have explored both pathways, the experiments with DMSO being antecedent the ones with AuNPs, because they do not require ad hoc synthesis of sulfur functionalized PCCs [3]. The outcome, though not straightforwardly applicable in the intracellular pH manipulation, is still quite interesting. Therefore, we report here the data we collected.
    Experimental design, materials and methods The effects of the DMSO on the intracellular uptake of HDNS were monitored by dosing them simultaneously to 3T3-NIH cells and subsequently probing them by infrared spectroscopy upon irradiation with UV-light. More in detail, NIH 3T3 Swiss Albino Mouse Fibroblast cells (ECACC Catalog number 85022108) were cultured directly on UV-transparent CaF2 windows in Dulbecco׳s Modified Eagle Medium (DMEM) with HCO3− (3.7g/L) and supplemented with 10% fetal bovine serum up to full coverage. Afterwards, the cells were incubated with 3mg HDNS and 2μL DMSO in 3mL in DMEM for 1h. The amount of DMSO was chosen as the minimum amount to allow an increased membrane permeability [4,5]. Afterwards the cell-coated window was transferred to the sample holder for liquids for collecting the infrared spectra, using a 12μm Mylar spacer. The experimental setup was the same as the one used afterwards for the gold coupled PCCs [3] (a Bruker IFS66/VS interferometer, in transmission mode with a resolution of 2cm−1). Infrared spectra were collected in the 3500–1000cm−1 range for reference spectra and 3000–1000cm−1 range for the cells. A few sequential infrared spectra of the cells were taken to verify their stability. Afterwards the samples were irradiated once for 1min with near-UV light, by using a deuterium discharge lamp (Acton Research Corporation) equipped with a band-pass FGUV11 filter (Thorlabs) in the 275–375nm. A few independent measurements were performed and they all provide similar outcome. Here we report two of them in Figs. 1 and 2. The data are treated with the OPUS software for vector normalization and offset correction. Finally, they are normalized by the first spectrum after irradiation and exported as ASCII files. The spectra are taken at intervals of 2min. Therefore, the whole datasets are taken in a time span of 30 (1st set) to 40min (2nd set). In the second data set a saturation level may be appreciated. The datasets are characterized by the arising of two bands which grow upon irradiation, one, rather large, centered at 2510cm−1 and the other one narrower centered at 1452cm−1. No contribution is found, instead, at 2343cm−1 the typical value for the intracellular CO2. The data, however, are largely reproducible, and are not related to dead cells, since we had previously verified that in our experimental set up they do not provide any spectral variation as a function of irradiation and of time. Furthermore, we made checks to identify the new arising features, by comparing them to those of DMSO and HDNS in DMSO upon irradiation. In Fig. 3 the difference spectra are reported of DMSO (panel (a)) and HDNS M in DMSO (panel (b)) before and 5min after UV irradiation. It can be observed that that DMSO is not affected by the irradiation. The PCC is obviously responsive to the irradiation and shows a number of positive and negative peaks that correspond to the breaking of the HDNS ester bond and the formation of a ketone and a sulfonic moiety, according to the mechanism illustrated in the inset of Fig. 3. In particular, the negative peaks at 1527cm−1 and 1346cm−1 are associated to the asymmetric and symmetric stretching of the NO2− group, the negative peak at 1235cm−1, to the sulfonic group. The positive peak at 1691cm−1 can be associated to the newly formed ketone, whereas the peaks at 1424cm−1, 1378cm−1 and 1271cm−1 are related to the cis and trans NO stretching [6]. The protons released in DMSO give rise to two new bands at 1652cm−1 and 3320cm−1 which can be associated to the bending and stretching of newly formed O–H+ interactions, as already observed for DMSO protonation by HCl [7]. The region around 2500cm−1 is free both for DMSO and for HDNS in DMSO. This rules out that in the datasets of the cells, the increase of features intensity upon irradiation is the direct observation of processes solely related to the molecules themselves. The interpretation is therefore to be found in processes which may affect the cells as effect of the proton release. There are many possible intracellular contributions to the feature at 1452cm−1, such as side chains and protonated side chains of several aminoacids and proteins as well as the Amide II band [8,9].