Alexa fluor 647 conjugated annexin 5

HEK293T cells stained Alexa Fluor 647-conjugated annexin V, 1:50 (BioLegend) and propidium iodide (1 µg/mL) in DMEM (Nacalai Tesque, 08459-64) with 10 mM HEPES on room temperature for 15 min. Samples were filtered through a 37 µm mesh prior to cytometry analysis.
Splenocytes were collected from adult and P9, P5 mice, and P5 interspecies chimeras by mashing the spleen between glass slides and chilling it in PBS(–). Splenocytes were stained with APC anti-mouse CD45 (1:50, Biolegend 100516), PE anti-mouse CD3ε (1:50, Biolegend 100308), Alexa Fluor 488 anti-mouse CD3ε (1:50, Biolegend 100321), APC anti-mouse CD4 (1:50, Biolegend 100516), PerCP anti-mouse CD8 (1:50, Biolegend 100732), APC anti-rat CD45 (1:50, Biolegend 202212), PE anti-rat CD3ε (1:50, Biolegend 201412), APC anti-rat CD4 (1:50, Invitrogen 17-0040-82), and PerCP anti-rat CD8 (1:50, Biolegend 201712) in 50 µL of 0.1% BSA/PBS at 4 °C for 30 min. The samples were resuspended in 500 µL of 0.1% BSA/PBS and filtered through a 37 µm mesh prior to flow cytometry analysis.
Flow cytometry was conducted using a BD Accuri C6 flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Becton Dickinson).

Trout leukocytes (~5 × 10⁵ cells) were seeded in 12-microwell plates, and co-cultured in 1 mL of L-15 + 10% FBS + 1% Anti-Anti (Gibco, Gaithersburg, MD, USA) with 0, 0.2, 1, and 2 mg/mL of UV-OxLDL at 15 °C. The leukocytes without OxLDL treatment were used as controls. After 2, 8, 24, and 48 hpi, the leukocytes were detached using a cell scraper and centrifuged at 200 g for 10 min at 4 °C. After removing the supernatant, the leukocytes were double-stained with 10 μg/mL of propidium iodide (PI; Sigma) and 0.5 μg/mL of Alexa Fluor^® 647-conjugated annexin-V (BioLegend, San Diego, CA, USA) in 100 μL L-15 + 10% FBS + 1% Anti-Anti for 15 min at RT. After adding 400 μL of annexin-V binding buffer (0.01 M HEPES, 0.14 M NaCl, and 2.5 mM CaCl₂), the fluorescence of PI and Alexa Fluor^® 647 in each cell was quantified using flow cytometry (Accuri C6™ Flow Cytometer, BD Biosciences, San Jose, CA, USA).