Non-radioactive labelling of calcium oxalate crystals for investigations of crystal-cell interactions and internalization

Abstract

The study of interactions between renal tubular cells and calcium oxalate (CaOx) crystals and their internalization was limited in the past due to lack of a simple method for visualization of CaOx crystals during such processes. We have developed non-radioactive techniques for efficiently labelling and imaging CaOx crystals in the study of crystal-cell interactions and internalization. A total of 12 ionic dyes, as well as AlexaFluor-488, FITC-conjugated IgG and Cy3-conjugated IgG were used to stain/label CaOx crystals. Thereafter, the crystals were incubated with MDCKcells for 48 h. The crystal images were obtained using light, phase-contrast, fluorescence, or laser-scanning confocal microscopy. The internalized CaOx crystals were finally quantified by flow cytometry. From 12 ionic dyes tested, CaOx monohydrate (COM) crystals were stainable only with CBB-R250 (blue) and Ponceau-S (red), whereas CaOx dihydrate (COD) crystals were stainable only with CBB-R250 (blue) and CBB-G250 (blue), which did not stain COM crystals but transformed them to COD. Additionally, only COM could be labelled and imaged with AlexaFluor-488 (green), FITC-conjugated IgG (green) and Cy3-conjugated IgG (red). Crystal-cell interactions (indicated by interrupted borders of crystals) and adhesion were successfully visualized under a light, phase-contrast, or fluorescence microscope. Moreover, laser-scanning confocal microscopic examination successfully identified internalized crystals, which could be quantified by flow cytometry. These non-radioactive techniques are very simple and effective for labelling and imaging COM and COD crystals for the study of crystal-cell interactions, adhesion and internalization, and will be very useful to investigate mechanisms of kidney stone formation.