Phagocytosis Assay and Flow Cytometry Analysis

A neutrophil infection assay was used to assess the phagocytic activity induced by the various Mmc strains (JCVI-syn1.0::mCh, JCVI-syn3A::mCh, and the add-back mutants, JCVI-syn3B::mCh-MMSYN1-0179–0181 and JCVI-syn3B::mCh-MMSYN1-0179–0186).²⁶ Initially, the phagocytic activity of dHL60 in coculture with JCVI-syn1.0::mCh and JCVI-syn3A::mCh was observed using a 3D Cell Explorer Microscope (Nanolive’s 3D Cell Explorer-fluo; Model CX-F). One mL of broth cultures of Mmc strains at the end of exponential phase of growth was spun down at 9000 RCF for 8 min at room temperature and suspended in100 μL of FBS (Sigma). We used this bacterial suspension to inoculate 350 μL of dHL60 cell suspension (8 × 10⁵ cells/mL) in RPMI media. The coculture was loaded in an μ-Dish 35 mm (ibidi) and kept at 37 °C and 5% CO₂ for video acquisition using STEVE (Nanolive) software. Images were captured every 1 min for 3 h.

In addition, Mmc infected and noninfected neutrophil-like cells were analyzed by flow cytometry to determine the rate of internalization or phagocytic index (PI) quantitatively. HL60 control samples and dHL60 cultures were spun down at 275g for 10 min in RT and suspended in filtered (0.1 μm) RPMI medium at a concentration of 6 × 10⁶ cells/mL. Viable cell counts were obtained using a trypan blue exclusion assay in a Countess Cell Counter (Invitrogen, CA). One mL of HL60 and dHL60 cells were used to suspend various Mmc strains prepared as described above and cocultured at 37 °C and 5% CO₂ for up to 210 min. HL60 and dHL60 cells and bacteria alone were used as control samples.

For flow cytometry, cells were evaluated using a BD FACS Aria II instrument (BD Biosciences), to investigate size, clustering patterns, and fluorescent levels. Our custom instrument is equipped with a forward scatter (FCS) photomultiplier tube to evaluate bacteria and small particles. To acquire signals from bacteria (0.2–0.4 μm diameter) and signals from the host cells (HL60 or dHL60; ∼12 μm diameter), FSC and side scatter (SSC) parameters (FSC PMT-A X SSC-A) were set in the logarithmic mode and used to set up the threshold signals. In addition to gate selection based on shape and size, we quantified the mCherry fluorescent label to the cell. mCherry signals using the PI-A parameter was acquired on logarithmic scale, and mean fluorescence intensity (MFI) was calculated within a host cell gated region, thus excluding cell debris region from control groups (media only) and unlabeled host cells. We set up our system to collect a total of 50,000 events per sample to develop uniformity across the samples. To further understand the kinetics of host–microbial interaction, all samples from each treatment were analyzed over time, at 30, 150, and 210 min of coculture. We obtained raw data in FCS format, which was evaluated by FlowJo v10.6.1 (BD Biosciences).

Phagocytic activity was expressed as phagocytic index (PI), and calculated using the following formula PI = (% phagocytic cells containing ≥1 Mmc) × (mean number of Mmc/phagocytic cell containing various Mmc).²⁷ Phagocytosis is the process by which a cell uses its plasma membrane to engulf a large particle or cell, such as a bacterium, giving rise to an internal compartment called the phagosome. Since in our experiments, Mmc expressed mCherry, the amount of fluorescence inside the HL60 or dHL60 cells was proxy for phagocytosis, which we calculated the PI for each treatment group. To account for possible fluorescent signal coming from the extracellular environment, we kept additional HL60 and dHL60 control cells on ice to metabolically deactivate host cells at similar time points of coculture. Signals from extracellular binding signals was eliminated during our analysis. Once host cells internalized the fluorescently labeled bacteria, we selected the gate and applied the same parameter to all samples, followed by MFI quantification and comparison across the groups.