Internalization of Nanofibrillated Cellulose in BEAS-2B Cells

To assess the potential internalization of NFC by BEAS-2B cells, NFC was stained with the Calcofluor White Stain (Merck KGaA, Darmstadt, Germany). The staining was performed on the same slides that were used for the scoring of the micronucleus frequency (for slide preparation, see below). At harvest, the cells were treated for 15 min at 25 °C with cellulase (8.7 µL/mL, Cellic CTec2, Novozymes, Bagesvaerd, Denmark) to remove excess NFC outside the cells. The procedure of the calcofluor staining has previously been described [35 (link)]. In brief, a drop of calcofluor was applied onto acridine orange-stained slides and incubated under a cover slip for 1 min. The sample was thereafter examined with a fluorescence microscope (ZEISS Axio Imager Z1, Carl Zeiss AG, Oberkochen, Germany), using DAPI/FITC/TRITC triple filter and 40x objective lens.