Fluorescent microspheres

All experiments were performed on an Elyra PS1 STORM/SIM microscope (Carl Zeiss Microscopy GmbH) equipped with a 100× objective (α Plan-Apochromat 100× 1.46 NA oil-immersion), a focus lock system and an EMCCD camera Andor iXon Ultra 897 (Andor Technologies). A LF488/561-A-000 beam splitter and a FF01–523/610–25 emission filter (Semrock) were used to record SEpH and mEos2 fluorescence. Antibodies against GFP labeled with Alexa 647 and anti-synaptotagmin antibodies labeled with Oyster 550 or Oyster 650 (Synaptic Systems) were detected using LF 405/488/561/635-A-000-ZHE and FF01-446/510/581/703-25 (Semrock). In dual color experiments, red and green channels were aligned with fluorescent microspheres (Molecular Probes) and the channel alignment module in Zen 2012 SP2 (black) software (Carl Zeiss Microscopy GmbH). We collected movies of labeled synaptotagmin sites in live neurons and only bright immobile fluorescent puncta were used to determine presynaptic terminals. Synaptic GABA_ARs were then identified from the overlap of synaptotagmin-rich presynaptic terminals with GFP-labeled γ subunit clusters. Cells were imaged in total internal reflection (TIRF) or highly inclined illumination mode in an enclosed chamber at RT, (32.0 ± 1.5)°C or (36.0 ± 1.5)°C.

NDP-MSH was purchased from Bachem (Bubendorf, Switzerland). Ultrapure LPS (E. coli, 0111:B4) and Pam₃CSK₄ were purchased from Invivogen (San Diego, CA, USA). Fluorescent microspheres (Molecular Probes, F8823) were kindly provided by Dr. Candolfi (INBIOMED, UBA-CONICET). Foetal bovine serum (FBS) was obtained from Natocor (Córdoba, Argentina). DMEM/F-12, DMEM, L-Glutamine and antibiotics were purchased from Invitrogen Life technologies (CA, USA). All other media and supplements were obtained from Sigma-Aldrich Corporation, unless specified otherwise.

Triplicate 1 ml samples for microbial cell enumeration using flow cytometry (FCM) were fixed with glutaraldehyde (2% final concentration), snap frozen and stored in liquid nitrogen on-board, prior to -80°C storage post-voyage. Prior to FCM analysis, samples were quick-thawed and divided to enable the separate enumeration of bacteria (200 μl) and autofluorescent picophytoplankton (800 μl). Samples for bacterial enumeration were stained with SYBR Green I [1:10,000] (Invitrogen Molecular Probes, United States), while picophytoplankton samples were analyzed unstained. For both sample types, 1 μm diameter fluorescent microspheres (Invitrogen Molecular Probes) were added as an internal reference (Marie et al., 1997 (link); Gasol and del Giorgio, 2000 (link)). Samples were analyzed using a Becton Dickinson LSR II flow cytometer (BD Biosciences), with bacteria discriminated according to SYBR Green fluorescence and side-scatter, while picophytoplankton populations were discriminated according to orange (phycoerthyrin) fluorescence, red (chlorophyll a) fluorescence and side-scatter (Seymour et al., 2007 (link)). All data were analyzed using Cell-Quest Pro software (BD Biosciences).

The phagocytic activity of monocytes was assessed using FITC-Dextran (Sigma Aldrich, 100 ng), FluoSpheres Red (580/605) Fluorescent Microspheres (Molecular Probes, 1 μm, 3.6 x 10⁷ microspheres/ml), and FITC-β-Ala-Amyloid β-Protein (1–42) (Bachem, 100 ng). Approximately 500,000 cells were resuspended in 500 μl of culture medium (MEM + 1 mg/ml BSA + 0.35 mg/ml NaHCO₃, pH 7.2) ± 1 μg/ml lipopolysaccharide (LPS) and incubated overnight at 37°C/5% CO₂, then centrifuged (300xg 10 min), resuspended in 100 μl FACS flow and analysed (BD FACS Calibur).
Cells were also characterized for their antigen expression. Following cultivation, cells were centrifuged at 300×g for 10 min, resuspended in 50 μl of FACS buffer (1% EDTA, 0.5% FCS, pH 7.1) containing primary antibodies against CD11b (1:25; BD, 557395), CD11c (1:25; Miltenyi, 130-091-842), CD14 (1:25; BD, 553739), CD45 (1:25; Miltenyi, 130-091-609), CD68 (1:5; Thermo Fisher Scientific, MA1-82739), F4/80 (1:10; Serotec/Biorad, MCA497FT), Ly6C (1:25; Miltenyi, 130-093-134), and major histocompatibility complex II (MHCII; 1:25, Miltenyi, 130-081-601) and incubated at 4°C for 30 min. Cells were subsequently washed, centrifuged and resuspended in 100 μl of FACS Flow and analyzed. All necessary IgG (IgG2a, 2b(k) and IgG1) controls were included.