Flow cytometer facs canto

Approximately 500,000 cells were seeded in 6-well plates and cultured for 24 h at 37 °C and 5% CO₂. Cells were washed with warm PBS and incubated with 50 nM EMI-137 for 1 h at 37 °C and 5% CO_2. Subsequently, cells were washed with ice-cold PBS three times prior to sample acquisition via FACS Canto flow cytometer (Becton Dickinson Immunocytometry Systems, Franklin Lakes, NJ, USA) with FACS Diva Software version 4.0.2. The obtained data were analysed using FlowJo software v7.6 (FlowJo, LLC, Ashland, OR, USA). Unstained controls were used to define gates and adjust fluorescence compensation.

H1975 cells were seeded in a 6-well plate at a seeding density of 500,000 cells/well, 24 h prior to performing internalisation assay. Cells were washed with warm PBS and incubated with 50 nM EMI-137 for 30 min or 1 h at either 4 °C or 37 °C, respectively. Subsequently, cells were washed with ice-cold PBS three times. To determine internalisation levels at 4 °C, surface-bound EMI-137 was removed by washing the cells with 50 mM of glycine prepared in 150 mM NaCl (Sigma) for 5 min followed by three washes with ice-cold PBS. To evaluate the internalisation dynamics of EMI-137, the incubation media (with EMI-137 at 4 °C for 30 min) were replaced with warm fresh media and incubated for 5, 15, and 30 min at 37 °C and 5% CO₂. Cells were then washed with glycine as described above. Samples were acquired with an FACS Canto flow cytometer (Becton Dickinson Immunocytometry Systems). Data were presented as mean Fluorescence Intensity (MFI). The internalised fraction was determined by subtracting the surface-bound EMI-137 from total EMI-137. The percentage of internalised fraction was calculated by dividing the amount of internalised EMI-137 by total-bound EMI-137 multiplied by 100%. Unstained controls were used to define gates and adjust fluorescence compensation.

For flow cytometry analysis, lungs were collected at day 1 after RSV or mock infection and digested with collagenase, as previously described (24 (link), 25 (link), 27 (link), 28 (link)). Cells were passed through nylon mesh to get a single cell suspension, and incubated with anti-FcγRIII/FcγRII mAb (anti-mouse CD16/CD32; BD Biosciences, San Diego, CA, USA) to reduce nonspecific binding, for 30 min at 4°C. After washing, cells were stained with the following anti-mouse antibodies: anti-F4/80 APC, anti-CD11b PerCP-Cy5.5, and anti-Ly6G FITC (for neutrophils). After incubation for 30 min at 4°C with the antibodies, cells were washed and then fixed in 200 µl of 1% paraformaldehyde in PBS. All the antibodies were purchased from BD Pharmingen, San Jose, CA, USA, except anti-F4/80 which was obtained from ebioscience, San Diego, CA, USA. Corresponding isotype Abs were used as controls. Cells were acquired with a FACS Canto flow cytometer equipped with Cell Quest software (both from Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Analysis was performed by using the FlowJo Software (Tree Star, NJ, USA).