Low melt agarose

Mice were euthanized and lungs were inflated with 2 mL dispase (Corning) and 0.5 mL 1% low melt agarose (Lonza) and allowed to sit covered with an ice pack for two minutes. Lungs were then removed from mouse and transferred to 1 mL dispase and incubated at room temperature for 45 minutes. Next, lungs were incubated in DMEM with DNase I (Sigma-Aldrich) at 95 U/mL and shaken for 10 minutes at room temperature. Lungs were homogenized in GentleMACS dissociator and red blood cells were lysed with ACK buffer. Cells were filtered to obtain a single cell suspension prior to staining. Cells were stained with Ghost Dye Red 780 (Tonbo), followed by the following antibodies against surface markers: CD45 (30-F11), podoplanin (clone 8.1.1), CD24 (M1/69), EpCAM (CD324, clone G8.8), MHCII (I-A/I-E, clone M5/114.15.2) (Biolegend), and CD31 (clone 390, BD Bioscience). Cell counts were obtained using AccuCheck counting beads (Thermofisher Scientific). Data were acquired on a BD LSRFortessa (Becton Dickinson, San Jose, CA).

The Tg (coro1a: EGFP; lyz: Dsred) line of fish was used in these experiments. When embryos reached 1 day post-fertilization (dpf), ~200 µM phenylthiourea (PTU; Sigma-Aldrich) was added to the culture water to inhibit the formation of pigmentation. At 2 dpf the fish larvae were injected with 1 nL of digested (200 μg/mL) or undigested CXCL8 (200 μg/mL), or with PBS into the hindbrain. Synthesized CXCL8 was digested with recombinant MMP9 as described above, whereas the undigested group was prepared by mixing CXCL8 with recombinant MMP9 just prior to injection. After 1.5 h, the larvae were fixed with 4% PFA for 2 h at room temperature. They were then washed with PBS, after which they were immunolabeled with anti-GFP and anti-RFP antibodies. The larvae were mounted in a glass capillary tube with 1% Low Melt agarose (50080; Lonza, Basel, Switzerland) and images of the head of the larvae were acquired using a Zeiss Lightsheet Z.1 3D microscope with a 5× water lens and the z-stack function. At the end of image acquisition, the left and right images collected were merged and deconvolution of each merged layer was performed, after which the images were exported using the maximum projection method and the numbers of neutrophils and macrophages in the hindbrain were quantified.

Mice were euthanized and lungs were inflated via intratrachial injection with 2 mL dispase (Corning) and 0.5 mL 1% low melt agarose (Lonza) and were incubated under an ice pack for 2 minutes. Lungs were removed, transferred to 1 mL dispase and incubated at room temperature for 45 minutes. Lungs were minced into small pieces, and transferred to DMEM with DNaseI (Sigma-Aldrich) 95 U/L and rocked for 10 minutes at room temperature. Lungs were homogenized in a GentleMACS dissociator, filtered through 70 μm filter mesh to generate a single cell suspension, RBCs were lysed and cells were stained for flow cytometry as described above.