Zombie green fixable viability kit

hTERT RPE-1 cells were treated as above, harvested, centrifuged and washed once in 1% bovine serum albumin (BSA) in 1XPBS. Cells were fixed in 100% ethanol (added dropwise while vortexing) and stored at +4 degrees overnight. Cells were centrifuged again and stained with staining solution -propidium iodide (20 μg/ml, Biotium), RNase A (1 mg/ml, Roche) in 1XPBS- for 3 h at room temperature (RT) prior to FACS analysis.
For assessing cell viability, hTERT RPE-1 cells were treated as above, harvested, centrifuged and resuspended in a Zombie Green™ Fixable Viability Kit (BioLegend) 1:100 in 1XPBS for at least 30 min at RT in the dark. Cells were centrifuged again and resuspended in 1XPBS.
In both cases, FACS was carried out on a FACS Celesta BD^TM (laser 488, filter 695/40 nm for propidium iodide; laser 488, filter 530/30 nm for Zombie Green). For each condition, at least 20,000 events were recorded, and data analysis was performed using the FlowJo software (v10.7.2). Cells were gated for singlets (as shown in Supplementary Fig. 1b) and then, for Zombie Green stained cells, FITC intensity was used to distinguish live from dead cells (as shown in Supplementary Fig. 3i), i.e. the percentage of live cells was obtained subtracting debris and Zombie-positive cells (i.e. dead cells) from the total.

Cells with membrane damage, which may have been caused by inhibitor treatments, were evaluated by the Zombie Green assay using the Zombie Green™ Fixable Viability Kit (Biolegend, USA). Cells were treated or not with the inhibitors following the manufacturer's instructions.

LAN-1 and KELLY cell lines were grown to 80% confluency and detached with Trypsin-EDTA solution. The SUPT1 GD2-positive and GD2-negative were both grown in suspension. For each cell line, 1.5×10⁶ cells were harvested and divided into three FACS tubes. Initially, a viability staining was performed by resuspending the cells in 100 μL of PBS-viability dye (Zombie Green Fixable Viability Kit, BioLegend Inc.) and incubating at room temperature for 30 minutes. One tube per cell line was used as negative control (unstained). For the remaining tubes, primary staining was performed by adding 10 nmol/L of either anti–GD2-IR800 or anti–GD2-IR12 conjugates in 50 μL of PBS. Secondary staining was performed by adding anti-IgG Alexa Fluor 647 (BioLegend Inc.) at a 1:2 ratio with the primary antibody. Both times, the cells were incubated at 4°C for 30 minutes and then washed twice with 1 mL of PBS. Samples were acquired on an LSRII flow cytometer (BD Biosciences). The analysis was performed using FlowJo software version 10.6.2 (TreeStar). The signal-to-background (SBR) was calculated as the median fluorescence intensity of the stained cells divided by the median fluorescence intensity of the unstained cells.

PBMCs of healthy individuals and UC patients were isolated. The cell pellet was resuspended in RPMI (Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 1×10⁶ cells/ml. Additionally, 500 µl RPMI with 10% FCS and 1% penicillin-streptomycin (Sigma-Aldrich, St Louis, MO, USA) were added to each well and sample. Wells containing PBMCs and RPMI or PBMCs, RPMI and anti-CD3 (1 µg/ml, BioLegend, San Diego, CA, USA) served as negative and positive controls, respectively. ritonavir was dissolved in 100% ethanol (10 mg/ml). For analysis of the effect of ritonavir, 100 µg/ml ritonavir (Sigma-Aldrich, Deisenhofen, Germany) was added. Cells were incubated for 72 h. The content of each well was centrifuged at 1400 g for 5 min. The pellet was resuspended in FACS buffer and labeled for flow cytometry.
To differentiate between live and dead cells, staining with Zombie Green™ Fixable Viability Kit (BioLegend) was performed according to the manufacturer's instructions.

Immunofluorescence assay was carried out as described previously [31 (link)]. The monoclonal antibody specific to V5 (Thermo Fisher Scientific, Waltham, MA, #R96025) and glycosomes were used at a 1:500 and 1:100 dilution, respectively [37 (link), 53 (link)].
For viability assay, T. brucei cells were labeled with the Zombie Green Fixable Viability Kit (BioLegend, San Diego, CA) and washed as stated by the manufacturer’s instructions. Cells were then fixed for 10 min in the presence of 4% paraformaldehyde in PBS and mounted on poly-lysine coated cover slips. Green and red cells were visualized with a fluorescence microscope and quantified with a hemocytometer. At least 200 cells were counted. This assay was performed twice in duplicate.