Cd11b pb

Splenocytes and thymic cells were isolated from WT and TRPV5 KO mice. Spleens were cut into small pieces and incubated in RPMI with collagenase (1 mg/ml, Roche) and DNase (0.1 mg/ml, Sigma) at 37°C with occasional stirring for 20 min to extract various innate immune cells. Single-cell suspensions were obtained and incubated with various antibodies to distinguish cell populations for 1 h at 4°C and washed three times with PBS. Splenocytes and thymic cells were immunostained with CD4–PE (1 µg/ml, Biolegend), CD8–APC (1 µg/ml, Biolegend), and CD3–Fluorescein isothiocyanate (FITC) (2.5 µg/ml, Invitrogen) to compare T-cell populations. Innate immune cell phenotyping of splenocytes consisted of immunostaining with CD11b–PB (2.5 µg/ml, Biolegend), CD11c–APC (1 µg/ml, Biolegend), CD64–PE (1 µg/ml, Biolegend), F4/80-APC/Cy7 (1 µg/ml, Biolegend), MHC Class II–BV605 (1 µg/ml, Biolegend), LY6C–AF700 (2 µg/ml, Biolegend), GR1–FITC (2 µg/ml, Biolegend), and NK1.1–PE/Cy7 (1 µg/ml, Biolegend). Cells were fixed after washing in 4% paraformaldehyde for 10 min at 4°C followed by three washes with PBS. Cells were analyzed on an LSR Fortessa, and data were analyzed using FlowJo (BD).

ASC cultures from early passages (1–6 population doublings) were harvested and resuspended in DMEM/F12 with 2% FBS. The cells were then centrifuged and suspended in cold PBS at a concentration of 10⁶ cells/100 μl. Cell aliquots were stained with monoclonal mouse anti-human antibodies against the following antigens: CD44-peridinin-chlorophyll-protein/cyanin 5.5, CD90-phycoerthyrin (PE), PE/Cy7-CD105, CD73-fluorescein isothiocyanate, CD34-Alexa Fluor 647, CD45-Pacific Blue (PB), CD11b-PB, and CD31-PB (BioLegend, San Diego, CA) for 30 minutes in the absence of light at 4°C. The cells that were stained with a single antibody coupled with a fluorescent dye were acquired for compensation purposes. After incubation, the cells were washed and resuspended in cold PBS. Flow cytometry was performed using a Becton Dickinson LSR II. A gate was set to include only the viable propidium iodide-negative cells. The number of cells staining positive for a given cell surface marker was determined by the percentage of cells present within an established gate. A minimum of 10,000 events were counted for each analysis.

NK cells were stained with fluorochrome-conjugated monoclonal antibodies against the following human surface antigens: CD56-PE-Cy7, CD16-APC-H7, CD3/CD14/CD19-PerCP-CY5.5, CD94/CD27/CD62L-FITC, NKG2C/NKp30/NKp46/NKG2D-APC, CD11b-PB, and NKG2A/NKp44/NKp80-PE (all purchased from Biolegend, San Diego, CA, USA). For intracellular staining, cells were washed, fixed, and permeabilized with Fix & Perm cell fixation and permeabilization kit (Invitrogen, Carlsbad, CA, USA) and stained with IL-4/TGF-β-FITC, TNF-α-PE, IL-10-APC, and IFN-γ-PB. Appropriate isotype controls were used. A minimum of 100,000 events were acquired using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with the FlowJo analysis software (Tree Star, Inc., Ashland, USA). Results were expressed as the percentage of positively stained cells in the NK cell gate.