Identification of apoptotic and necrotic cells

Cytotoxicity assay for identifying apoptotic and necrotic cells

A549 cells suspended in DMEM + 10% FBS were grown on 12-mm glass coverslips (VWR International BV, Amsterdam, the Netherlands) in 24-well plates (Corning®, Costar®, New York, USA) and challenged with 2x10⁶ spores/mL. Each condition was performed in duplicate and cells incubated with culture medium without spores was the negative control. After two hours the cells were washed three times with prewarmed culture medium, 380 µL medium without spores was added and this was again incubated at 37 ˚C for 4h, 12h, 18h and 24h. On the day of the assay a dye solution was freshly prepared by diluting 100 µg/mL acridine orange (AO; VWR International BV, Amsterdam, the Netherlands) and 100 µg/mL ethidium bromide (EB; Sigma-Aldrich, Zwijndrecht, the Netherlands) in DPBS. After the desired incubation time the cells were washed twice with prewarmed DPBS and 380 µL of the dye solution was added to the cells. This was incubated at RT for one minute before the coverslips were retrieved and transferred onto a 10 µL drop of FluorSave™ on a glass slide and pressed gently to a tissue. The glass slides were examined under a fluorescent microscope within 15 minutes. 

Fluorescence microscopy and image analysis

Confocal images for the association and internalization experiments were acquired with a Zeiss LSM700 confocal laser scanning microscope (AxioObserver Z1 stand) using Plan-Apochromat 63x/1.40 oil immersion lens. For excitation of dTomato from AF293.1 a 555 nm laser has been used and fluorescence emission was detected using the spectral band 562-600 nm. For excitation of FITC-antibodies a 488 nm laser has been used and the emission fluorescence was detected with the 490-520 nm spectral band. For excitation of CFW and Hoechst a 405 nm laser has been used and the emission fluorescence was detected with the spectral band 490-520 nm.

At least 10 random images were analyzed per condition. Association was expressed as the conidia/cell-ratio. Internalization was expressed as percentage of internalized conidia, using the following equation: . 

Fluorescence microscopy images for the cytotoxicity experiments were acquired with an Axioskop 2 plus at a 20X magnification. The microscope had four filters: DAPI, FITC, TRITC and Cy5, corresponding with the filter set 90 HE ms. The excitation and emission wavelengths of these filters and the AO/EB staining were retrieved from the manufacturers website and are shown in Table 2. Although both AO and EB were visible with the FITC filter, the TRITC filter predominantly showed EB, allowing for a better distinction of the two dyes. Thus, imaging of EB and AO was performed with a FITC filter and for single detection of EB a TRITC filter was used. Per condition at least 1000 cells were analyzed from at least three different pictures.

All images from the association, internalization and cytotoxicity assay experiments were analysed with the Fiji image processing package of ImageJ (www.fiji.sc).