Lymphocyte Isolation and Incubation

Lymphocytes were extracted following a previous protocol established in our lab from controls (Romay-Tallon et al., 2018). These extracted lymphocytes were then resuspended in RPMI 1640 medium (#11875093, Thermoscientific, Waltham, MA) diluted with 10% phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ ; pH 7.4) [w/v] and 1% streptomycin and incubated first with either medium or 1 mM CORT, and then with varying concentrations of reelin (0.5 nM, 1 nM, 5 nM) and ketamine (10 nM, 50 nM, 100 nM, 250 nM) in the medium specified above. 1% [w/v] paraformaldehyde was added for 5 min to the solution to fix the lymphocytes, and then was centrifuged in 1:1 PBS (two times, 1,000 x g, 10 min) to rinse. 1 mM of CORT was used as it is equivalent to the 40mg/kg injected in our rat model and has paralleled changes that we have observed in patients with depression (Romay-Tallon et al., 2018).

After incubation, lymphocytes were incubated for 10 min at 4°C in a 100 ml solution of 3% rat immunoglobulin G (IgG) diluted in PBS with 1% BSA [w/v] (PBS + BSA), then for 24 h at 4°C in rabbit anti-serotonin transporter (SERT) antibody (#AB9322, Millipore Sigma, Burlington, MA), diluted 1:100 in PBS+BSA. After washing 3x in PBS for 10 min, samples were incubated with goat anti-rabbit secondary antibody (#ab175471, abcam, Cambridge, UK) diluted 1:250 in PBS+BSA and conjugated with Alexa Fluor 568 in the dark for 1 h at room temperature and then for 10 min in Hoechst diluted 1:1,000 in PBS. After 3× 10 min washes, samples were extended onto slides and cover-slipped with Citifluor-Mount Solution (Electron Microscopy Science) and stored at −20°C until analysis. Through compound microscopy, lymphocytes were analyzed using ImageJ to quantify the number and size of SERT clusters.