Tali cellular analysis slide

The msn-mCherry D. melanogaster strain was used to assay expression of msn. This strain carries a transgenic construct containing the msn-F9 enhancer upstream of the mCherry red fluorescent protein [51 (link)]. Second instar msn-mCherry larvae were exposed to either AsDen or L. boulardi for a 72-h period as described above, with 3 biological replicates for each infection condition. Host hemocytes were isolated 72 hpi and added to a Tali Cellular Analysis Slide (Invitrogen). Hemocytes were allowed to adhere for 30 min and then cell number, size, perimeter, circularity, and red fluorescence intensity were measured using a Tali Image-Based Cytometer (Invitrogen). For each replicate, we imaged 20 fields of cells, with an average of 717.4 cells per field, and a range of 194 to 1455 cells for a total of 32,176 hemocytes from L. boulardi-infected larvae and 53,908 hemocytes from AsDen-infected larvae. Cytometer data were filtered to only include single cells using the Tali software count function and size-gating, prior to further analysis.

The analysis of cell death was performed using a Tali Image-based cytometer and a Tali Apoprosis kit (both from Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, following trypsinization, the suspended cells were resuspended in Annexin binding buffer at a concentration of approximately 5×10⁵ to 5×10⁶. Subsequently, to each 100 μl of sample, 5 μl of Annexin V Alexa Fluor 488 were added, mixed and incubated at room temperature in the dark for 20 min. The cells were then centrifuged and resuspended in 100 μl of Annexin binding buffer. After the addition of 1 μl of propidium iodide (PI) to each sample, the cells were incubated at room temperature in the dark for 3 min. A total of 25 μl of stained cells were loaded into a Tali Cellular Analysis Slide (Invitrogen Life Technologies). The data were analyzed using FCS Express Research Edition software (Ver4.03; De Novo Software, Los Angeles, CA, USA) on the assumption that viable cells are both Annexin V Alexa Fluor 488- and PI-negative cells, cells that are in early apoptosis are Annexin V Alexa Fluor 488-positive and PI-negative, cells that are in late apoptosis are both Annexin V Alexa Fluor 488- and PI-positive, whereas necrotic cells are Annexin V Alexa Fluor 488-negative and PI-positive.

HaCaT cells and HGFs were pre-treated with ULH-002 at different concentrations for 2 h. Then, the cells were irradiated with UVA (32 J/cm²). At 2 h after UVA irradiation, cellular ROS generation in cells was detected with the CellROX^® Orange or Green Reagent (Thermo Fisher Scientific) according to the manufacturer’s recommended protocol. ROS production in cells was observed by the EVOS^® FL Cell Imaging System (Thermo Fisher Scientific). To qualitatively analyze the ROS production, HaCaT cells and HGFs were detached from the culture substratum-surface by 0.025 w/v% trypsin–0.01% EDTA solution and then suspended in PBS (-) at a concentration of 1 × 10⁶ cells/mL. In addition, 25 µL of the cell suspension was infused into a Tali™ Cellular Analysis Slide (T10794, Thermo Fisher Scientific, Tokyo) and analyzed by the Tali™ Image Cytometer (Thermo Fisher Scientific, Tokyo, Japan).