The protocol reported here describes a simple, easy, fast and reproducible method aimed to know the geometric parameters of living cells based on confocal laser scanning microscopy combined with 3D reconstruction software. Briefly, the method is based on intrinsic fluorescence properties of acridine orange (AO), a molecule taken up by living adherent cells. Dual binding of AO to either DNA or RNA allows complete staining. When combined with confocal microscopy, 3D software can be used for in vivo living cell reconstruction. Beside the purpose that we intend here, a fast and easy system for cell volume determination, the protocol is an easy approach to study changes in morphology during cellular processes such as cell differentiation. Novel therapeutic approaches would require some knowledge about how these drugs enter into cells/tissues. For this purpose fast and accurate in vivo cell volume determinations such as the method reported here, in combination with analytical methods, would allow estimating intracellular concentrations of compounds and might be further employed for finding out whether any new drug can reach the effective concentration inside its cellular target. Furthermore this protocol with minimal adjustments will permit the determination of morphometric parameters in vivo in different types of adherent cells.
A straightforward and common analytical method for α-tocopherol (αT) determination in various biological samples, including plasma, red blood cells (RBC), tissues and cultured cell lines, was developed and validated, using a reverse phase-chromatographic method (RP-HPLC). Even though many chromatographic methods for αT determination have been reported, most of them require readjustment when applied to different types of samples. Thus, an effective and simple method for αT determination in different biological matrices is still necessary, specifically for translational research. This method was applied using a C18 column (250 × 4.6 mm, 5 µm particle size) under isocratic elution with MeOH:ACN:H2 O (90:9:1 v/v/v) at a flow rate of 1 mL/min and detected using photodiode array at 293 nm. Linearity (r >0.9997) was observed for standard calibration with inter- and intraday variation of standard <4%. Lower limits of detection and quantification for αT in this assay were 0.091 and 0.305 µg/mL respectively. Validation proved the method to be selective, linear, accurate and precise. The method was successfully applied in great variety of biological samples, that is, human and mouse plasma, RBCs, murine tissues and human/mouse/rat cultured cell lines. More importantly, a single protocol of extraction and detection can be applied, making this method very convenient for standardization of different types of samples.
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