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We are committed to excellence in innovation and research and we want to take advantage of SPAchip® possibilities to explore other areas in the biomedical sector, such as Delivery of molecules for therapeutic applications or diagnostics to detect biomarkers in clinical samples.

SPAchip® size (3x3x1 µm3) and geometry have been optimized to avoid cytotoxicity while yielding an optimal uptake ratio (it represents less than 1% of a standard cell volume), although internalization is cell type dependent. We suggest adding a 2:1 SPAchip®:cell ratio at the measurement time point, but optimization may be required. By adding the suggested ratio to the culture, most of the cells show only 1 SPAchip® in the cell cytosol, although there can be cells which have uptaken more than 1 SPAchip®. Greatly increasing the SPAchip®:cell ratio will not offer a significantly higher value of SPAchip®-positive cells, however there will most likely be a higher distribution of cells with more than 1 internalized SPAchip®.

At A4Cell we have validated SPAchip® use in more than 20 different cell lines and primary cultures, including some of the most frequent cells used in screening (e.g. HeLa, CHO, HEK 293T, Hepatocytes). Adherent and suspension cells have also been validated, both from primary origin and immortalized lines. Until now, all cell lines tested have been proven to internalize SPAchips in their cytosol and our team is continuously working on expanding the applications portfolio including different cell models.

SPAchip® internalization rates are variable across cell lines. An average of 25-40% cells with positive SPAchip® internalization has been observed in different cell lines, ranging from >95% internalization in macrophages to ~10% in 3T3 cell. These values should be enough to perform statistical analyses between study conditions in classical multiwell plate setups.

Regarding SPAchip® use in vivo, we have not data yet, but it will be provided soon.

SPAchip® intracellular distribution is always cytosolic and no specific subcellular location has been identified. Adherent cells usually show SPAchip® near the plasma membrane in 2D cultures, likely due to their flat shape. SPAchip® have never been seen inside the cell nucleus, nor being ejected to the extracellular medium after internalization.

Remaining SPAchip® in the extracellular medium can be subject to cell internalization after incubation. However, a unique feature of SPAchip® devices is given by the fact that these extracellular SPAchip® can be identified as a different population, either by fluorescence microscopy or flow cytometry, to simultaneously obtain intra and extracellular values of the analyte of interest.

SPAchip® are continuously detecting changes in the analyte levels in the cytosol offering dynamic, real-time measurements by changes in the fluorescence intensity. Compared to traditional end-point assays, SPAchip® are advantageous because preparation of multiple samples to analyze different time points become unnecessary, thus saving time and resources. Additionally, the possibility of monitoring samples over time allows to keep track of drug responses, differentiation processes or cell trajectories, which could not be precisely assessed otherwise.SPAchip® are made of silicon oxide, an inert material, which allows to maintain cellular viability offering absence of cytotoxicity. Fluorescent probes used as sensors in SPAchip® are covalently bond to the surface to avoid degradation and release to the intracellular environment. The small quantity of fluorescent probes also reduces the interaction with the whole analyte levels increasing the physiological resemblance in the cell model.

SPAchip® Assay Buffer contains a saline solution with physiological pH and osmolarity to maintain the conditions of standard culture media. It is provided to dissolve the solid film where Assay SPAchip® are contained and wash them to remove it. Additionally, SPAchip® solution is needed to be further diluted in cell culture media to avoid any possible interaction with the culture, as stated in the SPAchip® kit protocol.

Lack of cytotoxicity offered by SPAchip® devices make them suitable for their use in cell assays that require extended culture time, such as delayed drug responses, differentiation processes, bioproduction, etc. Our team has been able to keep cells in culture for 1 month after SPAchip® addition, while keep cell viability and SPAchip® fluorescence signal.

On the contrary, the minimum suggested cell incubation with SPAchip® is overnight. SPAchip® uptake has been seen to happen after 1h in some cell types, however an overnight incubation is recommended to obtain optimal results.

SPAchip® are solid devices, thus they do not follow cell growth over time. For this reason, upon cell division only one of the daughter cells keep the SPAchip® in its cytosol, hence SPAchip®-to-cell ratio should be designed according to the expected cell number/confluency.

SPAchip® kits have been designed to work with fluorescence signals as the primary readout, where a change in the SPAchip® fluorescence intensity correlates with an increase/decrease in the analyte levels. Specific control conditions need to be included in the assay to obtain quantitative data.

The main instruments to analyze SPAchip® readouts are fluorescence microscopes and flow cytometers. Microplate readers are not a suitable option for SPAchip® analysis due to their whole well scanning process, as it does not allow resolving such small volumes.

Excitation and emission wavelengths are specified in each SPAchip® kit protocol.

Video recording can be performed to analyze fast occurring analyte fluxes in the cell cytoplasm with sub-second frequency when imaging SPAchip® samples

We have CytoCHECK SPAchip® single-detection kits for measuring pH and Calcium in standard GFP/FITC channel.

We will be expanding our portfolio of products soon to offer novel applications and fluorescence wavelengths

To identify single-detection SPAchip® where the fluorescence signal covers the whole device surface (3×3 um2) a magnification of at least 20x is required to identify the objects in the subcellular environment. Higher magnification will yield improved results.

Image analysis software and pipelines are suggested to be used in order to identify different objects and subcellular structures. If you require assistance, please contact A4Cell Team.

As any other fluorophore, SPAchip® can be subject of photobleaching effect. However, our devices have been optimized to suffer a mild decay in the fluorescence signal (<20%) after continuous and intensive laser exposure (1h, 13 cycles), with all calibration conditions maintaining significant differences in intensity.

SPAchip® can be used with any other fluorescent readouts, including stainings, engineered cell lines, cell painting approaches, etc. Excitation and emission wavelengths need to be considered to avoid undesired signal overlap. Please contact A4Cell if you need support to design your panel.

We are committed to excellence in innovation and research and we want to take advantage of SPAchip® possibilities to explore other areas in the biomedical sector, such as Delivery of molecules for therapeutic applications or diagnostics to detect biomarkers in clinical samples.