Cell Culture and Assays

Human acute promyelocytic leukemia (HL-60) cells and mouse H22 hepatoma cells from American Type Culture Collection (ATCC) were purchased from the Chinese Academy of Sciences and were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO₂ and 95% air at 37°C. To obtain N1-like neutrophils (N1), HL-60 cells were seeded at 2 × 10⁵ cells/ml in medium supplemented with 10 μM all-trans retinoic acid (named HL-60-N1) or 0.5 μM DOX (named HL-60-N1-D) for 7 days in a flask (Almzaiel et al., 2013; Li et al., 2018), changing the medium after 3 days. To obtain N2-like neutrophils (N2), HL-60-N1 cells were further stimulated with 100 ng/ml TGF-β1 for 3 days (Fridlender et al., 2009; Sagiv et al., 2015) and were named HL-60-N2. To collect cell-conditioned media, N1 or N2 cells were cultured in serum-free medium for 24 h, centrifuged to remove cells, further filtered to remove debris for supernatant collection, and named N1 cell-conditioned media (N1-CD) and N2 cell-conditioned media (N2-CD), respectively. The supernatant levels of IFN-γ, IL-2, PD-L1, IL-6, IL-10, and TGF-β1 were determined using ELISA kits according to the manufacturer's protocols. The results were calculated from linear curves obtained using the Quantikine kit standards.

For proliferation assays, HL-60 cells at 1 × 10⁵ cells/ml were seeded in a 96-well plate and treated with N1 or N2 cell-conditioned media, DOX or berberine alone or in combination for 48 h. N1 or N2 cells were also treated with DOX or berberine alone or in combination for 48 h, and living cells were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, according to our previous method (Liu et al., 2015). For differentiation analysis, HL-60 cells at 3 × 10⁵ cells/ml were seeded in six-well plates and treated with N1 or N2 cell-conditioned media, DOX or berberine alone or in combination for 5 days (changing the medium after 3 days), and neutrophil differentiation was assessed by Giemsa staining and microscopic NBT assay according to the previously described methods (Guo et al., 2017; Li et al., 2019). For the morphological assessment, cells were analyzed by a laser holographic cell imaging and analysis system (HoloMonitor M4, Phiab, Sweden). For phagocytic ability, intracellular phagocytosis of Escherichia coli was detected by Giemsa staining (Li et al., 2019). For autophagic analysis, cells were stained using FITC-conjugated anti-LC3-B or anti-p62 antibodies. For apoptotic analysis, the binding of ANXV-FITC to phosphatidylserine was measured by an automated cell counter and analysis system (Nexcelom Cellometer X2, Nexcelom, USA). For reactive oxygen species (ROS) measurement, the intracellular fluorescence of DCFH-DA was detected by a fluorescence spectrophotometer (Hitachi F-4600, Japan). For the time-lapse migration assay (Patel et al., 2018), cells were placed onto a motorized stage and observed with a laser holographic cell imaging and analysis system (HoloMonitor M4, Phiab, Sweden). A 20× objective was used to capture images during the course of the time-lapse. Images were captured every 15 s over the course of 30 min from at least four different fields of view.

Immunofluorescence was performed according to a previously described method (Guo et al., 2017). After overnight incubation with primary antibodies (CD66b, CD133, CD309, and PD-L1), slides were incubated with FITC-conjugated goat anti-mouse IgG for 30 min. The semi-quantitative immunofluorescence score was calculated by using the intensity score and proportion score by excluding the primary antibody and IgG matched serum as positive and negative controls, respectively.