Live single cell functional phenotyping in droplet nano-liter reactors.

Materials Rat FITC-anti-CD86 antibody [GL1] was obtained from Abcam and used at concentration of 1 μg/10 6 cells. Biotin-anti-IL-6 Ab (504601) for bead conjugation was obtained from BioLegend and FITC-anti-IL-6 Ab (11-7061-81) for IL-6 detection was obtained from eBioscience and used at concentration of 1 μg/mL. Microfluidic device fabrication Microfluidic flow chambers were fabricated using soft lithography at the MGH/CEM Facility. Negative photo resist SU-8 2100 (MicroChem, Newton, MA) was deposited onto clean silicon wafers to a thickness of 150 μm, and patterned by exposure to UV light through a transparency photomask (CAD/Art Services, Bandon, OR). The Sylgard 184 poly(dimethylsiloxane) (PDMS) (Dow Corning, Midland, MI) was mixed with crosslinker (ratio 10:1), poured onto the photoresist patterns, degassed thoroughly and cured for 12 hours at 75°C. Next, we peeled the PDMS devices off the wafer and bonded to glass slides after oxygen-plasma activation of both surfaces. The device is composed of a droplet forming nozzle and a storage array for 10 3 droplets. To improve the wetting of the channels with mineral oil in the presence (1%, w/w) of the surfactant (span80), prior to the experiments the microfluidic channels were treated with Pico-Sur™ 2 (Dolomite Microfluidics, UK) by filling the channels with the solution as received and then flushing them with air. Tygon Micro Bore PVC Tubing 100f, 0.010" ID, 0.030" OD, 0.010" Wall (Small Parts Inc, FL, USA) were connected to the channels and to the syringes. We used 1 ml syringes to load the fluids into the devices, while syringe pumps (Harvard Apparatus, USA) controlled the flow rates. Microsphere sensors preparation ProActive® Streptavidin Coated Microspheres (10 μm) (Bang Laboratories Inc., USA) were conjugated with the biotinylatedanti-mouse IL6 (Abcam®, MA, USA) according to the manufacturer's protocol. Unbounded active sites were blocked with StarlingBlock™ (thermo Scientific, USA) for one hour. Next, we washed the beads with the Phosphate Buffered Saline (PBS) (Sigma, USA), diluted to the final concentration 0.5 mg ml −1 , and stored at 4°C.

Cell isolation and stimulation 6–8 week old female

C57BL/6J mice were obtained from the Jackson Laboratories.

Bone marrow dendritic cells

(BMDCs) were collected from femora and tibiae and plated on non-tissue culture treated plastic dishes in RPMI medium (Gibco, Carlsbad, CA, Invitrogen, Carlsbad, CA), supplemented with 10% FBS, L-glutamine, penicillin/streptomycin, MEM non-essential amino acids, HEPES, sodium pyruvate, β-mercaptoethanol, and GM-CSF (15 ng/mL; Peprotech, Rocky Hill, NJ). At day 7 floating cells were collected and used as GM-CSF derived dendritic cells. Cells were stimulated with LPS (rough, ultra-pure E. coli K12 strain LPS, 100 ng/ml; Invitrogen, San Diego, CA).

Image analysis

Fluorescence images of droplets were captured on a Zeiss 200 Axiovert microscope using an AxioCAM MRm digital camera. For the cell studies, fluorescence signals were captured separately using appropriate filter sets for FITC. Image processing and analysis was conducted using ImageJ software.

Materials Rat FITC-anti-CD86 antibody [GL1] was obtained from Abcam and used at concentration of 1 μg/10 6 cells. Biotin-anti-IL-6 Ab (504601) for bead conjugation was obtained from BioLegend and FITC-anti-IL-6 Ab (11-7061-81) for IL-6 detection was obtained from eBioscience and used at concentration of 1 μg/mL.