Atcc ccl240

HL60 cells purchased from ATCC (ATCC CCL-240) were differentiated with 1.25% dimethyl sulfoxide (DMSO) (Sigma-Aldrich) in RPMI for 4 days (65 (link)). Differentiated HL60 cells were harvested and checked for >65% viability before starting the assay in accordance with a well-characterized protocol (66 (link)). In a 96-well plate (Corning), 50 μl containing a total of ~1 ×10³ CFU of mid-exponential phase GBS, normalized in HBC buffer (1× HBSS, 0.5% BSA, and 2.2 mM CaCl₂, pH 7.2), were added. Fifty microliters of either 20% normal human serum (Complement Tech) or 20% heat-killed human serum (56°C for >30 min) in HBC buffer was added to each well and incubated at 37°C, with shaking, for 15 min. HL60 cells were preincubated with either 20 μM cytochalasin D (Sigma-Aldrich) or DMSO (Sigma-Aldrich) vehicle control for 15 min, with rotation, before 100 μl of HL60 cells (~1 × 10⁵) was added to each well and incubated at 37°C with shaking (MOI of ~0.01) as described previously (66 (link), 67 (link)). After 3 hours, 10 μl was removed, serial diluted, and plated on THB agar for enumeration. Assays were performed in biological triplicate with technical duplicate per replicate. Percent survival was calculated as the ratio of CFU in normal serum versus CFU in heat-killed serum, and no HL60 control wells were set to 100% as described in (68 (link)).

Free and SFN-PPh were tested using macrophage cells obtained from differentiation of the human acute myeloid leukemia cell line HL-60 (ATCC^® CCL-240™, American Type Culture Collection, Rockville, MD, USA) as the selected in vitro model. Cells were maintained in CCM consisting of RPMI-1640 (Biowest, Nuaillé, France), supplemented with 10% fetal bovine serum (GIBCO Invitrogen, Paisley, UK) and 1% penicillin/streptomycin (GIBCO), at 37 °C with 5% CO₂ before differentiation.
All in vitro assays were performed after passage number 5 and before passage number 20 with cells growing at an exponential ratio. Cells were plated at 1 × 10⁵ cells/well (96-well plates) or 1 × 10⁶ cells/well (12-well plates) in CCM containing 10 ng·mL⁻¹ phorbol myristate acetate (PMA) (Sigma Chemical Co., St. Louis, MO, USA) for a period of 24 h to allow differentiation. Cells were then rested in CCM without PMA for another 24 h prior to the addition of the different treatments.

Streptococcus equi subsp. zooepidemicus ATCC35246 (SEZ ATCC35246) was isolated from a dead pig in Sichuan Province, China. The bifA gene deletion mutant ΔBif and the complement strain CBif were constructed in a previous study [7 (link)]. Neutrophil-like human leukemia cell line HL60 (ATCC^® CCL-240™) and HEK293T cells (ATCC^® CRL-3216™) were purchased from American Type Culture Collection (ATCC). Bacteria were cultured in Todd Hewitt Broth (THB) medium. The HL60 cells were maintained in IMDM medium with 20% fetal bovine serum (Gibco, Grand Island, NY, USA) in a 37 °C incubator containing 5% CO₂. The HL60 cells were induced to differentiated HL60 (dHL60) cells by 1.3% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) in IMDM for five days (Supplementary Figure S1, see Appendix A) [24 (link)]. Primary mouse neutrophils were isolated from SPF ICR mice and kept in RPMI 1640 medium (Gibco, Grand Island, NY, USA) [25 (link)].