DDAO

This plan only provides a guide, please modify it to meet your specific needs.
1. Solution preparation
(1) Stock solution: Dissolve DDAO in DMSO to prepare a stock solution with a concentration of 5mM.
Note: After unused stock solution is aliquoted, store it at -20°C or -80°C in the dark to avoid repeated freezing and thawing.
 
(2) Working solution: Before the formal experiment, dilute the stock solution with a suitable buffer (such as serum-free medium or PBS) to the required working concentration, such as 1μM.
Note: Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration by yourself. The working solution must be prepared and used immediately.
 
2. Using DDAO to study the phagocytic effect of macrophages on cancer cells^[1](from the literature, for reference only)
(1) 0.8×10⁶ RAW264.5 cells or 1×10⁶ THP1 cells were seeded into 6-well plates.
(2) After incubation for 24h, the RAW264.7 cells were treated with 0.1μg/mL lipopolysaccharide (LPS) for 24h to activate them. THP1 cells were differentiated by treatment with 40nM phorbol 12-myristate 13-acetate (PMA) for 48h.
(3) Activated macrophages were stained with DiO (Cat. No. GC19515) at a final concentration of 40nM in culture medium for 20min and then rinsed three times with culture medium.
(4) Cancer cells were labeled with 1μM DDAO (5mM stock, DMSO, stored at −20°C) in PBS at 37°C for 15min and then washed with PBS containing 1% FBS.
(5) DDAO-labeled target cells (1×10⁶) were added to DiO-stained macrophages (Mφ) and incubated at 37°C in a final volume of 2mL for 2h.
(6) After incubation, Mφ and target cells were harvested by washing three times with EDTA-PBS and then digested with 0.25% trypsin.
(7) Flow cytometry was used to evaluate double-labeled cells (DIO⁺/DDAO⁺), which represent cancer cells engulfed by mature Mφ.
 
3. Using DDAO to study B cell proliferation^[2](from the literature, for reference only)
(1) B cells were labeled with 1μM DDAO and cultured in complete RPMI supplemented with 4μg/mL CD40L, 100ng/mL BAFF, 1μg/mL anti-IgM or 2.5μg/mL CpG.
(2) After 3 days of culture, DDAO dilution (and mCherry expression in CRISPR-targeted B cells) was measured by flow cytometry.

References:
[1] Candas-Green D, Xie B, Huang J, et al. Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells[J]. Nature communications, 2020, 11(1): 4591.
[2]Malinova D, Wasim L, Newman R, et al. Endophilin A2 regulates B?cell endocytosis and is required for germinal center and humoral responses[J]. EMBO reports, 2021, 22(9): e51328.