Latex bead

Phagocytosis of inert particles (latex beads, 1.1 µm diameter, Sigma-Aldrich) was assayed in phagocytes (isolated blood neutrophils) following a method previously described (57 (link)). Neutrophils adjusted to 10⁶ cells/mL were incubated on migration inhibition factor plates (Kartell, Noviglio, Italy) for 30 min at 37°C in a humidified atmosphere. The adherent monolayers obtained were washed with prewarmed PBS solution, and then 200 µL of Hank’s solution and 20 µL of latex beads (1.1 µm diluted to 1% PBS, Sigma-Aldrich) were added. After 30 min of incubation under the same conditions, the plates were washed, fixed with methanol (50%), and stained with Giemsa’s solution (Sigma-Aldrich). The number of particles ingested by 100 neutrophils was counted using an immersion objective (×100) and this was expressed as phagocytic index, while the number of ingesting neutrophils per 100 neutrophils was expressed as phagocytic efficiency.

Hemocytes from L. vannamei at 48 h post dsRNA injection were washed with 2× Leibovitz’s L-15 medium (Gibco, USA) and mixed with latex beads (Merck, Germany), fluorescein isothiocyanate (FITC)-labled V. parahaemolyticus or S. aureus at a 1:100 ratio of cells/particles. After incubation at 28°C for 1 h, hemocytes were detected using Accuri C6 flow cytometer (BD, USA) for the signals of FITC and the forward scatter (FSC) values of cell size. The threshold of FSC was determined by detection of free latex beads, FITC-labeled V. parahaemolyticus or S. aureus, and the fluorescence boundary was set based on detection of the self-fluorescence of untreated hemocytes (Figure S1).

To analyze the phagocytosis activity of Znfx1^–/– BMDMs, cells were infected with H37Rv-RFP or incubated with FITC-conjugated Latex beads (catalog L4530, Merck Millipore). Cells were then collected and analyzed with flow cytometry.
To detect the numbers of macrophages in various tissues of Znfx1^–/– mice, single cells obtained from the spleens, the lungs, and the LNs of WT or Znfx1^–/– mice were incubated in 1% FBS-PBS containing the following antibodies in the dark at 4°C for 30 minutes before flow cytometry analysis: PerCP/Cyanine5.5 anti-mouse/human CD11b (M1/70; BioLegend), Violet Fluor 450–F4/80 (BM8.1; Tonbo Bioscience), and PE-Ly6c (HK1.4; BioLegend).
To assess the activation of BMDMs infected with H37Rv, cells were collected with trypsinization and incubated in 1% FBS-PBS containing the following antibodies at 4°C in the dark for 30 minutes: FITC-CD80 (16-10A1; Thermo Fisher Scientific), APC-CD86 (GL-1; Tonbo Bioscience), PE-Cy7–MHC-II (M5/114.15.2; Invitrogen), eFluor 450–CD11b (M1/70; Tonbo Bioscience), and PE-CD206 (C068C2; BioLegend). Cells were then centrifuged at 300g at 4°C for 5 minutes, fixed with 4% paraformaldehyde, and analyzed.
Cells were detected using Attune NxT flow cytometry (Thermo Fisher Scientific), and the data were analyzed with FlowJo 10 software (BD Biosciences).

BV2 cells were inoculated on coverslips and stimulated as described above. Erythrocytes were isolated from newly drawn mouse blood with Ficoll (Merck Millipore, Germany). Briefly, the same amounts of PBS and blood were mixed, gently added to Ficoll in a centrifuge tube, and centrifuged at 2000 rpm for 25 minutes with minimum acceleration and deceleration. The erythrocytes were washed twice with PBS before labeling with DiI (Beyotime, China), a fluorescence indicator for the cytomembrane. The labeled erythrocytes were added to the BV2 cells in the plate at a ratio of 10 : 1. After 2 hours of phagocytosis, undigested erythrocytes were dislodged with Red Blood Cell Lysis Buffer (Beyotime, China), and the BV2 cells were labeled with Actin-Tracker Green (Beyotime, China) according to the product manual. The proportion of BV2 cells containing ingested erythrocytes was quantified using fluorescence microscopy. For flow cytometry (FCM), latex beads (Merck Millipore, Germany) with red fluorescence were used for the phagocytosis assay, and the cells were harvested to quantify the phagocytic ability after 2 hours of phagocytosis. The gating strategy of phagocytosis is shown in Sup. Fig 1 B and the data were analyzed using FlowJo software.