Draq7

A total volume of 100 µL of each set of cell suspension was incubated in the dark with 2 µL of propidium iodide (PI) (Invitrogen) (1mg/mL) or 0.5 µL of DRAQ7 (Biostatus) (0.3 mM) before measurement by flow cytometry using a Cytoflex (Beckham Coulter), in the Flow Cytometry Service at CINBIO (University of Vigo). PI and DRAQ7 are membrane-impermeable dyes that specifically stain the nuclei of dead cells, as they are excluded from viable cells. Upon excitation by specific lasers (560 nm for PI and 640 nm for DRAQ7), these dyes emit fluorescence at wavelengths of 617 nm (PI) and 665 nm (DRAQ7) when bound to double-stranded DNA. This fluorescence is detected by the corresponding channels and correlates with the percentage of dead cells. To select the optimal medium and CPA, and prior to the analysis of the acclimation effect, PI was used for the initial quantification of the viability after the freezing/thawing process for fresh and directly thawed cells. For the following flow cytometry viability experiments described, DRAQ7 was used instead of PI because of its compatibility to be visualized at the same time in the fluorescent microscopic analyses. To further analyze the effects of cell acclimation after thawing, all three experimental groups (fresh, directly thawed, and thawed acclimated cells) were analyzed. For the thawed acclimated set, cells of each thawed vial were resuspended after thawing in a total of 300 µL of the selected media, seeded in an 8-well chamber slide, and kept at 15°C for 18h before processing. The negative fluorescence threshold and possible autofluorescence emission of hemocytes were previously established and tested using unlabeled cells. This experiment was repeated at least three times.
Fluorescence microscopy was performed to visualize cell morphology and viability following the freezing and thawing protocol. A batch of cells was directly thawed following the optimal protocol previously described and seeded into an 8-well plate in a final volume of 300 µL of MAS media. Fresh cells were seeded under identical conditions. To analyze the possible impacts of the freezing/thawing protocol on the cell morphology, the last experimental group (thawed acclimated cells) was also seeded and kept in MAS media for a total of 18h before the analysis and maintained at 15°C before the image analysis. The morphology of fresh, directly thawed, and thawed acclimated cells was analyzed under a Thunder fluorescent inverted microscope (Leica). Images were recorded for 4h every 10 minutes and analyzed to detect morphology differences for the different cell types. This experiment was repeated three times.

Expression of recombinant TCR in the 5KC reporter system was performed as previously described^{80 (link)}. To construct TCR- expressing vectors, a fragment containing a human T cell receptor alpha variable gene (TRAV) variable segment, one with the mouse TRAC*01 gene fused to the viral P2A sequence and one with a human T cell receptor beta chain variable segment (TRBV) were synthesised (Genewiz, Azenta) with overlaps and joined to the backbone of an Murine Stem Cell Virus (MSCV)- based vector containing a murine TRAV signal peptide and the murine TRBC1 gene by Gibson assembly^{81 (link)}. Vectors were introduced into 5KC hybridoma cells containing the 8xNFAT-ZsG reporter, muthCD4 along with either LSSmOrange (LO), blue fluorescent protein (BFP) or E2 Crimson (CR) or combinations thereof, by transfection into Phoenix cells and subsequent transduction. All cell lines were generous gifts from Professor Maki Nakayama and have been described^{82 (link)}. CD3⁺ (TCR expressing) live cells were FACS-sorted (ARIAIII, BD) and further expanded in culture medium. For antigen stimulation, 150,000 APCs (the CD3^--, CD4^--, CD8^-- live cell fraction of PBMCs) were incubated with 75ug/ml GAD (T cell-GAD, 10-4508029-01, Dyamid) or, as positive and negative controls, 2.5ug/ml hamster NALE anti-mouse CD3e (clonotype 145 2C11) or Dyamid Control, respectively, for 4 hours. 20,000 recombinant TCR- expressing 5KC cells were then added for overnight incubation in a total volume of 200uL in culture medium. Draq7 (BioStatus) was then added and the expression of the ZsG- reporter was acquired on a LSRFortessa (BD) and analysed with FlowJo v10.8.

Cells were washed and resuspended in Annexin V binding buffer (BD Biosciences, Franklin Lakes, NJ) and stained with Annexin V (BD Biosciences) and DRAQ7™ (Biostatus Ltd., Shepshed, UK). Subsequently, stained cells were analyzed with an LSRFortessa or Accuri flow cytometer (BD Biosciences) for assessment of cell death. Data analysis was performed using the FlowJo software (TreeStar Inc., Ashland, OR).

Cell death in MCA205 stimulated with class I and class II ferroptosis inducers was measured as described before^{80 (link)}. Briefly, cells were seeded at 10000 cells/well in 96-well adherent plates and the next day they were treated with cell death inducers. All the inhibitors were added 4 hours before cell death induction. Cell death rate was calculated based on the fluorescence intensity of SytoxGreen^® (1 µM). Fluorescence was measured by Fluostar Omega (BMG Labtech GmbH). For PS exposure analysis, cells were collected, washed in 1x Annexin Binding Buffer and stained with Annexin V-APC (#BMS306APC/100, eBioscience, 1:100) or Annexin-FITC (#BMS306FI, ThermoFisher, 1:100), 1.25 µM SytoxBlue® (#S11348, Molecular Probes,) and alternatively 1 µg/ml 7-AAD (#A-1310, Molecular Probes) or 3 µM DRAQ7 (#DR71000, Biostatus UK) and cell death was measured with an LSRII flow cytometer and the data were analyzed using FlowJo 10.2 software.