High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells.

2-channel ratiometric TIRF microscopy

TIRF microscopy was performed on a custom-built microscope with excitation at 473 nm from a diode-pumped solid-state laser (Blues 50, Cobolt, Solna, Sweden) delivered via a single mode optical fiber (SMF) and a rotatable mirror to adjust the TIRF angle. Excitation is delivered into the back-port of an inverted epifluorescence microscope (IX71, Olympus, Tokyo, Japan) equipped with a X60, 1.45NA oil-immersion TIRF objective. Fluorescence is collected on an electron-multiplying CCD camera (EMCCD) (iXon, Andor, Belfast, UK) in the range 500-593 nm and 600–680 nm using a 2-channel imager (Dual-View, Optical Insights) ( Fig. 1 ). Fluorescence above 680 nm was not collected due to high background fluorescence (Data not shown). Data were processed using custom software (LabVIEW, National Instruments, Austin, TX). Calculated GP values ( Equation 1 ) for each pixel were then pseudo-coloured (from high membrane order – red, to low membrane order – blue; see colour bars for Figs. 2 - 5 ). The images were merged with the fluorescence intensity image such that the result displays both order and structural information. The sample was maintained at 37°C using a stage and objective heater (PeCon, Erbach, Germany). Quantification of image data was performed by measuring average GP values in the central region (defined from the centre to half way to the synapse edge) and the remaining peripheral region. Equation 1 GP = I 500 − 593 − I 600 − 680 I 500 − 593 + I 600 − 680 Confocal microscopy Single-photon excitation confocal microscopy was undertaken using an inverted, epi-fluorescence laser-scanning confocal microscope (TCS SP5, Leica Microsystems Ltd., Wetzlar, Germany) with a 63×, 1.25NA oil-immersion objective lens. Excitation was at 488 nm from an argon-ion laser. Fluorescence detection was in the wavelength bands 500–580 nm and 620–750 nm using internal photomultiplier tubes. Z-slices were acquired every 700 nm. GP values were calculated as for TIRF images. 3D reconstructions and en face views were generated using Volocity software (Improvision, Waltham, MA). Fluorescence lifetime imaging For FLIM, fluorescence was collected in the range 540-750 nm using a PMT and TCSPC electronics (SPC-830, Becker and Hickl GmbH, Berlin, Germany) with single-photon excitation at 460 nm from a frequency-doubled Ti:Sapphire laser (Tsunami, Spectra-Physics, Mountain View, CA). Fluorescence decays were fitted to a single-exponential function and pseudo-coloured as for the GP images (see SI Text for experimental details). Optical tweezing Cell and IS orientation by optical tweezing was performed using an infrared (980 nm) optical trapping beam as previously described ( 28 ). Confocal fluorescence imaging was then performed in the same way as for the 3D confocal microscopy.

Sample preparation Jurkat

T cells (clone E6.1) and Raji B cells were cultured in RPMI1640 medium (PAA, Pasching, Austria) supplemented with 10% foetal calf serum (FCS) at 37°C in a 5% CO 2 atmosphere. Raji B cells were loaded (or not) with the superantigen Staphylococcal enterotoxin E (SEE) for 90 min at 37°C.

Human CD4 +

T cells were isolated using a MACS kit for negative selection (Miltenyi Ltd) after separation of peripheral blood mononuclear cells on Ficoll gradient (GE Healthcare). One hour prior to imaging, the cells were resuspended in HEPES-buffered salt saline (HBSS) supplemented with 1mM Ca 2+ , 2mM Mg 2+ , 1% gelatine and 5 μM di-4-ANEPPDHQ (Invitrogen, Carlsbad, CA). The use of serum-free medium containing gelatine dramatically reduces the fluorescence background from residual dye which would otherwise swamp the red wavelength channel ( Figure S1 ). Coverslip chambers (Lab-Tek, Brendale, Australia) coated with poly- L -lysine followed by 10 μg/ml antibodies to CD3 (OKT3 mAb) in phosphate-buffer saline (PBS) and blocked by incubation at 37°C with PBS supplemented with 1% gelatin for 30 minutes were used for activatory surface stimulation of T cells. Supported planar bilayers with GPI-anchored forms of I-E k and ICAM-1 were prepared in FCS2 flow cells (Bioptechs, Butler,USA) and used for stimulation of AND T cells as described before ( 2 ). The I-E k molecules on the bilayers were loaded with 100 μM MCC88-103 peptide in citrate buffer for 48 hr. The density of the GPI-anchored proteins in the bilayers was: I-E k 20 sites/μm 2 and ICAM-1 150 sites/μm 2 . (see SI Text for details). For ordered phase disruption using 7KC, 15 mg/ml 7KC and cholesterol (both from Avanti Lipids, Alabaster, AL) in ethanol were combined in the ratio 2:1. Over 30 mins, these were then added to a solution of 50 mg/ml methyl-β-cyclodextrin (MβCD; Sigma-Aldrich, St.Louis, MO) in PBS at 80°C to a final sterol concentration of 1.5 mg/ml. 15 μl of the lipid solution were then added to 1 ml of cell medium containing 5×10 5 cells at 37°C for 30 minutes.

Supplementary Material Supplemental Fig 1 Supplemental text Video 1 Video 2 Video 3