Dear Yanrong Zhang!

Here is the answer, I hope proper! GOOD LUCK in experiments!

Sincerely yours,

Dr. Bratko Filipič

Email: [email protected]

__________________________________________-

Isolation of primary mouse lung macrophages

The use of animals in this study was approved by the Institutional Animal Care and Use Committee of North Carolina State University (Raleigh, NC, USA). CD-1 mice were purchased from Charles River (Wilmington, MA, USA) at 6–10 weeks old. The animals were heavily sedated with ketamine/xylazine (100 mg/kg and 10 mg/kg body weight, respectively), administered i.p. and exsanguinated by cutting the left renal artery and abdominal aorta. The abdomen was exposed carefully by cutting through the ribcage, avoiding the lungs. Red blood cells were flushed using a 10-ml syringe, and a 21-gauge needle was used to inject 5–10 ml 0.9% sterile NaCl through the right ventricle of the heart. The trachea was exposed surgically and a 20-gauge catheter inserted. The lungs then were filled with 3 ml dispase through the catheter, followed immediately by instillation of 0.45 ml 1% low-melt agarose, and then the thorax was covered with crushed ice for 2 min to solidify the agarose. The trachea was sealed with surgical silk and the heart and lungs removed and rinsed with 5 ml sterile PBS. The lungs were transferred to 2 ml dispase in a sterile culture tube and then placed in a 37°C water bath for 30 min, after which, they were placed in 7 ml freshly prepared DMEM with 100 μl 0.7% DNase I type II, put in a 60-mm Petri dish, and teased apart carefully and gently swirled for 5–10 min to release cells. The mixture was then filtered through a 100-μ sterile cell filter into a 50-ml Falcon tube prior to gentle centrifugation at 200 g for 12 min at 4°C. The supernatant was aspirated and the cell pellet resuspended in 10 ml DMEM + 10% FBS and incubated for 30 min at 37°C in 5% CO2. Macrophages stuck to the plastic dish, whereas unattached cells were washed away with sterile PBS, and fresh medium was then added to the cells. To ensure that the cell suspension preparation was enriched with lung macrophages, differential staining with Diff-Quick, followed by counting under a light microscope, was performed. Viability of isolated lung macrophages was assessed using a Cellometer with trypan blue dye exclusion; macrophage viability was >90%.

Transwell migration assay

Migration assays were performed in 6.5 mm Transwell plates with 8 μm pore inserts. The upper side of one insert was thinly coated for 1 h with rat tail type I collagen. J774A.1 cells or primary murine macrophages (1×105 cells) were resuspended in 100 μl chemotaxis buffer (RPMI 1640 plus 0.02% BSA). Cells were added to the upper chamber and 600 μl migration medium, with or without chemotactic factors: MCP-1 (25, 50, or 100 ng/ml), PMA (10, 50, or 100 nM), or C5a (5, 10, or 20 ng/ml) added to the lower chamber. Cells were allowed to migrate through the insert membrane for 3 h at 37°C under a 5% CO2 atmosphere. In some experiments, cells were first pretreated with MANS or RNS peptide (25–100 μM) or PBS for 15 min at 37°C. The inserts were then washed with PBS, and non migrating cells remaining on the upper surface of the insert were removed with a cotton swab. The migrated cells on the insert were fixed, stained with Diff-Quick, and mounted on glass slides. Migration was measured visually by counting using a light microscope at 40× magnification. The mean number of cells in 10 randomly chosen fields was calculated for each treatment. A migration index was calculated by dividing the number of cells that migrated in response to the chemokine by the number of cells that migrated randomly (RPMI/0.02% BSA) with a reference index >1, indicating chemotaxis.

Thank you very much Dr. Bratko Filipič!

Yanrong

Dear Yanrong Zhang!

Its my pleasure!

Sincerely yours!

Dr. Bratko Filipič

Email: [email protected]