Cd14 fitc staining

5 × 10⁶ leukocytes were washed in PBS and then incubated in 500 µL PBS with 10% goat serum (S-1000, Vector Laboratories) for 30 min to block non-specific binding. The cells were resuspended in 100 µL PBS with 10% goat serum for 1 h at room temperature with primary or without (control) rabbit antibodies against SLC5A5/NIS (SAB2102220, Sigma) or SLC26A4/PENDRIN (MBS9215961, MyBioSource). The cells were washed and then stained for 30 min with secondary F(ab′)2 goat anti-rabbit (Invitrogen, A21246) at a 1:100 dilution and CD45 Krome orange (Beckman Coulter, A96416) at a 1:20 dilution at room temperature. Other experiments included CD14 FITC staining (BD Pharmingen, 555397) to identify monocytes. Cells were washed, resuspended in IsoFlow sheath fluid (Beckman Coulter), and then loaded onto BD FACSCanto II where 25,000 events were collected in the lymphocyte gate. The resulting data and the median fluorescence intensity (MFI) were analyzed utilizing FlowJo software.

The study protocols were approved by the Clinical Research Ethics Committee of our institution (Comitè Ètic d’Investigació Clínica, HuGTiP, Ref. CEIC: PI⁻13-011), and conformed to the principles outlined in the Declaration of Helsinki.
Peripheral blood monocytes were obtained from leucocyte residues from healthy donors provided by the Blood and Tissue Bank (Barcelona, Spain). Succinctly, peripheral blood mononuclear cells were depleted of CD3⁺ cells using the RosetteSep^TM Human CD3 Depletion Cocktail (StemCell Technologies, Seattle, WA) during a Ficoll-Paque density centrifugation (GE Healthcare, Uppsala, Sweden) and monocytes were then positively selected by MagniSort™ Human CD14 Positive Selection Kit (eBioscience, San Diego, CA), as recommended by the manufacturer. Monocytes were assessed for purity by CD14-FITC staining (BD, San Jose, CA), obtaining >95% CD14⁺ cells analysed in a Canto II flow cytometer (BD).
Culture medium comprised RPMI 1640 medium supplemented with 100 µg/ml streptomycin, 100 IU/ml penicillin and 5% heat-inactivated human AB serum (H4522, Sigma, St Louis, MO). Cells were counted using PerfectCount Microspheres (Cytognos, Salamanca, Spain) and viability was >94% as determined by 7-AAD exclusion by flow cytometry (FACSCanto II, BD).