β mercaptoethylamine mea

The dSTORM images were acquired using an inverted wide-field fluorescence microscope (IX-71; Olympus). For excitation a 641 nm diode laser (Cube 640–100 C, Coherent, Cleanup 640/10, Chroma) was focused onto the back focal plane of the oil-immersion objective (60 × , NA 1.45; Olympus). Emission light was separated from the illumination light using a dichroic mirror (635rpc, Chroma) and spectrally filtered by a bandpass filter (Em01-R442/514/647–25; Semrock). Images were recorded with an EMCCD (IXON DU897, Andor). Resulting pixelsize for data analysis was measured as 128 nm. For each dSTORM measurement, at least 15,000 images with an exposure time of 20 ms and irradiation intensities of ~2 kW/cm2 were recorded by HILO (highly inclined and laminated optical sheet) illumination. Experiments were performed in PBS-based photoswitching buffer containing 100 mM β-mercaptoethylamine (MEA, Sigma-Aldrich) for Cy5 and an oxygen scavenger system (2% (w/v) glucose, 4 U/ml glucose oxidase and 80 U/ml catalase) or only in PBS for Cy5B and HMSiR, adjusted to pH 7.4. Image reconstruction was performed using rapidSTORM3.3^{61 (link)}.

For the dSTORM measurements the sample was excited with a 642 nm diode laser (100 mW output power). A series of 4000 TIRF images at a resolution of 70 nm/pixel was recorded for a single sample position. Images were recorded using an exposure time of 20 ms. The used imaging buffer contained a glucose oxidase based oxygen scavenger in combination with 90 mM β-mercaptoethylamine (MEA; Sigma-Aldrich, Taufkirchen, Germany).

Daudi B cells were washed in PBS once then stained in 2% FBS/PBS at a concentration of 1 × 10⁶ cells/ml with 10 μg/ml of Alexa Fluor® 647 labeled pinatuzumab IgG and 2 μg/ml Alexa Fluor® 488 labeled anti-human IgM Fab fragment for 20 min at 4°C. Cells were washed 2 times with PBS and resuspended in PBS. Cells were incubated at 37°C for 5 min before injecting in FSC2 chambers preheated to 37°C and allowed to spread for 10 min. Chambers were gently flushed with PBS to wash unbound cells and cells were fixed with 4% PFA and 0.2% glutaraldehyde in PBS for 40 min at room temperature. Chambers were washed 3 times with PBS. Prior to image acquisition, chambers were incubated with dSTORM imaging buffer containing 0.1 M β-mercaptoethylamine (MEA, Sigma-Aldrich), 0.5 mg/ml glucose oxidase, 40 μg/ml catalase, and 10% glucose in PBS. For dual-color dSTORM, samples were incubated in PBS containing 0.1 M MEA, 3% (v/v) OxyFlourTM (Oxyrase Inc.), 20% (v/v) of sodium DL-lactase solution (L1375, Sigma-Aldrich) adjusted to pH ~8.3. Fiducial markers (100 nm Tetraspeck Fluorescent Microspheres, Invitrogen) were added to buffer and allowed to settle for 5 min prior to imaging.