Real-time single-cell imaging of protein secretion.

Reagents The DuoSet ELISA Development

System was purchased from R&D Systems (Minneapolis, MN, USA), and the set of capture and detection antibodies was used for sandwich immunoassays for human IL-1β (DY201). Human IL-6 monoclonal antibody (clone 6708; MAB206) and human IL-6 biotinylated affinity-purified polyclonal antibody (BAF206) were also purchased from R&D Systems. Adenosine 5′-triphosphate disodium salt hydrate (ATP, A7699) and lipopolysaccharides from Escherichia coli 055:B5 (LPS, L4524) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipidure BL802, a water-soluble polymer of 2-methacryloyloxy ethyl phosphorylcholine, was purchased from NOF Corporation (Tokyo, Japan). Calcein AM (C3099) and SYTOX Blue nucleic acid stain (S11348) were purchased from Life Technologies (Carlsbad, CA, USA). CF660R streptavidin was purchased from Biotium (29040; Hayward, CA, USA). Dimethyl pimelimidate-2HCl (DMP, 21666) was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Foetal bovine serum (FBS, s1560) was purchased from Biowest (Nuaillé, France).

Cell culture

The incubation of cells was performed in a CO 2 incubator at 37°C in a humidified atmosphere with 5% CO 2 , unless otherwise indicated. For the isolation of human peripheral blood monocytes, 20 mL of venous blood was drawn from a healthy donor after obtaining institutional approval of the ethical committee of the Kyoto University Hospital, in accordance with Declaration of Helsinki. Monocytes from different donors were used for different experiments. The cell fraction was separated with Lymphoprep (Axis-Shield, Dundee, UK) according to the manufacturer's instructions. CD14 + monocytes were sequentially purified by MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) with a negative selection reagent (i.e. monocyte isolation kit II) and a positive selection reagent (i.e. CD14 microbeads). The isolated monocytes were incubated overnight in RPMI medium containing 10% FBS. Optical arrangement All measurements were performed with a completely automated inverted microscope (ECLIPSE Ti-E; Nikon, Tokyo, Japan) equipped with a high NA 60× objective lens (TIRF 60 × H; NA, 1.49; Nikon). The microscope was customized to introduce external laser beams (635 nm; Radius 635-25; Coherent, Santa Clara, CA, USA) at the indicated incident angle to actualize TIRF illumination. The following sets of excitation (Ex) and emission (Em) filters and a dichroic mirror (DM) were used with a high-pressure xenon lamp: for SYTOX, Ex = FF01-448/20-25, Em = FF01-482/25-25, and DM = Di02-R442-25x36; and for calcein, Ex = FF02-472/30-25, Em = FF01-520/35-25, and DM = FF495-Di03-25x36. The following Ex and Em filters and DM were used with the 632 nm laser for the CF660R dye: Ex = FF02-628/40-25, Em = FF01-692/40-25, and DM = FF660-Di02-25x36. These optical filters were purchased from Semrock (Rochester, NY, USA). Each image was projected on an EM-CCD camera (ImagEM C9100-13; Hamamatsu Photonics K.K., Sizuoka, Japan) through a 0.7× lens (C-0.7x DXM Relay Lens; Nikon). A stage-top incubator (ONICS; Tokai Hit Co., Shizuoka, Japan) was used to control temperature, humidity, and gas concentration. MWA chips MWA chips were prepared from PDMS and microscopic-grade coverslips. The PDMS MWA chip was prepared as follows: A thin PDMS sheet, consisting of a 50 × 50 array of 50-μm or 70-μm through-holes with 100-μm centre-to-centre spacing and 80-μm thickness, was purchased from Fluidware Technologies (Saitama, Japan). The PDMS sheet and a glass slide were permanently bonded together after the contact surfaces between them were plasma-treated (SEDE-PFA; Meiwafosis, Tokyo, Japan). The bare glass surfaces that now functioned as the bottom of the microwells after bonding were aminated with Vecatabond Reagent (SP-1800; Vector Laboratories, Burlingame, CA, USA). The chip was mounted on a coverslip holder (Attofluor cell chamber, A7819; Life Technologies) adjacent to another PDMS block (Sylgard184; Dow Corning Toray, Tokyo, Japan) with an 8-mm diameter through-hole that served as a reservoir well. The interstices between the PDMS-glass chip and the PDMS block were filled with uncured PDMS. The integrated chip was baked at 130°C for 3 h. A 100-μL mixture of capture antibodies (100 μg/mL) and dimethyl pimelimidate-2HCl (DMP; 7 mg/mL) was loaded onto the aminated glass surface to fix the capture antibody. The surface was blocked with monoethanolamine (0.1 M, pH 8.2) and Lipidure reagent (0.2% [w/v]), and stored at 4°C until use.

Preparation of detection medium

Detection antibody labelled with biotin was coupled with CF-labelled streptavidin at 1:10 molar ratios at room temperature in the dark for 3 h. Unoccupied sites on streptavidin were blocked with excess dPEG4-biotin acid (10199; Quanta BioDesign, Ltd., Powell, OH, USA). Unconjugated streptavidin and dPEG4-biotin were removed by ultrafiltration (Amicon Ultra-0.5, 100 kDa; Merck Millipore, Billerica, MA, USA). The detection media contained prepared CF-labelled detection antibody (30 nM), BSA (2.5% [w/v]), and the indicated combination of the following additives (with the final concentrations): SYTOX (0.8 μM), glycine (5 mM), LPS (1 μg/mL), and/or ATP (5 mM).

Handling and treatment of cells

Monocytes were stimulated with LPS for 3 h and incubated with 0.4 μM Calcein AM for 15 min before IL-1β secretion analysis on a PDMS MWA chip. The cells were washed with fresh medium and recovered from the bottom of 96-well plates by gentle pipetting. Approximately 2,000 cells were introduced into an MWA chip and allowed to settle by gravity for 10 min in a CO 2 incubator. Next, the medium was replaced with the detection medium containing SYTOX, glycine, and LPS. The cells were first monitored for 20 min before ATP stimulation; after ATP administration, monitoring was resumed during the 10-min incubation period. The medium was then replaced with fresh detection medium containing SYTOX, glycine, and LPS. Measurements were then resumed and continued for 4 h. Time-resolved monitoring of cytokine secretion from stimulated monocytes Ten positions were selected in order to include as many single cell-containing microwells as possible. Monocytes in the selected position of microwells were monitored at 1-min intervals by multichannel microscopy, i.e. DIA images, epifluorescence images for calcein and SYTOX, and TIRF images for CF660R for cytokine secretion at controlled temperature and gas concentrations. The MFIs of each microwell at each time point were measured with the NIS Elements imaging software. Transition time estimation of calcein, SYTOX and IL-1β signals obtained from LPS/ATP stimulated monocytes The transition time of calcein disappearance, SYTOX influx, and IL-1β release were detected by data analysis software (Origin 8.6; OriginLab Co., Northampton, MA, USA) by using the following equations: for calcein disappearance, for SYTOX influx, and for IL-1β release, where I(t) represents intensity, I 0 represents the background, m represents intensity drift, I 1 represents amplitude, t 0 represents transition time, and τ represents the time constant of exponential decay for each fluorescence signal. The subscripts c , s , and i of each parameter indicate calcein, SYTOX, and IL-1β labelling, respectively. The time constant of IL-1β ( τ i ) was determined from a supplementary experiment with recombinant IL-1β ( Fig. 2 ). The IL-1β secretion dataset was fitted by equation (3) .

Supplementary Material Supplementary Information movie 1 Supplementary Information movie 2 Supplementary Information Supplementary information