Flow cytometry (FC) is a procedure that allows multiparameter analysis of the suspended cell component in an individual, cell-by-cell manner, through its physicochemical characteristics, identifying the expression of cellular proteins and any cellular component or function that can be labeled with a fluorochrome. Flow cytometry allows you to measure the different parameters of a cell. These are divided into nuclear parameters, cytoplasmic parameters, surface parameters and extracellular parameters. Phytoplanktonic microalgae, both marine and freshwater, are mostly unicellular and rarely measure more than a few tens of micrometers in diameter, making them perfect candidates for easy analysis of their integrity of cytoplasmic and mitochondrial membranes using commercial flow cytometry equipment [21 (link),22 (link),23 (link),24 ,25 (link),26 ,27 (link)]. Part of the biological material of the microalgae Scenedesmus sp. thawed by each treatment (DMSO 5%, DMSO 10%, MET 5%, MET 10%, Control) was used in parallel to perform the respective flow cytometry studies and thus determine the integrity of the cytoplasmic and mitochondrial membrane. These biological samples were processed in the laboratories of the Cellular Immunology and Immunogenetics (GICG) group of the Cytometry Unit of the Medical Research Institute of the University of Antioquia, Medellín.
The straws cryopreserved with each treatment were thawed in a serological water bath (Memmert, WNB 7–45, Baviera, Schwabach, Germany) at a constant temperature of 35 °C for 90 s. Once thawed, they were deposited in 5 mL plastic tubes. Subsequently, 100 μL was taken with a micropipette (Transferpette®, CE704174, Baden-Württemberg, Wertheim, Germany) of the microalgae Scenedesmus sp. and deposited in another pipe, to which was added 400 μL of an enveloping solution (isotonic solution) plus 50 μL of a solution containing fluorochromes; 3,3′-dihexyloxocarbocyanine (DiOC6 (3)) (70 nm) + propidium iodide (PI) (Sigma, St. Louis, MI, USA) at a rate of 2 μg/mL with a micropipette (Transferpette®, CE704174, Baden-Württemberg, Wertheim, Germany. This was the case for each treatment. Subsequently, the corresponding tests were performed with a FACS CANTO II CYTOMETER (BD LSRFortessa™ San José, California, USA) by excitation of a 488 nm laser and fluorescence detection at 530/30 nm and 670 nm for DiOC6 (3) and PI, respectively. In the same way, fresh samples not cryopreserved were taken as controls, and the same management was given.
The straws cryopreserved with each treatment were thawed in a serological water bath (Memmert, WNB 7–45, Baviera, Schwabach, Germany) at a constant temperature of 35 °C for 90 s. Once thawed, they were deposited in 5 mL plastic tubes. Subsequently, 100 μL was taken with a micropipette (Transferpette®, CE704174, Baden-Württemberg, Wertheim, Germany) of the microalgae Scenedesmus sp. and deposited in another pipe, to which was added 400 μL of an enveloping solution (isotonic solution) plus 50 μL of a solution containing fluorochromes; 3,3′-dihexyloxocarbocyanine (DiOC6 (3)) (70 nm) + propidium iodide (PI) (Sigma, St. Louis, MI, USA) at a rate of 2 μg/mL with a micropipette (Transferpette®, CE704174, Baden-Württemberg, Wertheim, Germany. This was the case for each treatment. Subsequently, the corresponding tests were performed with a FACS CANTO II CYTOMETER (BD LSRFortessa™ San José, California, USA) by excitation of a 488 nm laser and fluorescence detection at 530/30 nm and 670 nm for DiOC6 (3) and PI, respectively. In the same way, fresh samples not cryopreserved were taken as controls, and the same management was given.
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