Yellow-green fluorescent beads (Thermo Fisher Scientific, Cat. # F8803, 100 nm diameter) were used for experimental FWHM measurements for iSIM. Beads were diluted from the stock concentration 1:1,300 (1:100 in distilled water and 1:13 in ethanol) and spread over cleaned glass cover slips. After air-drying for 5 minutes, coverslips were washed twice in distilled water to remove unattached beads. After air-drying again, beads were mounted in oil (Cargille, Cat. # 16241) onto glass slides and sealed with nail polish.
Yellow green fluorescent beads
Manufactured by Thermo Fisher Scientific
Yellow-green fluorescent beads are small, spherical particles that emit yellow-green fluorescent light when exposed to a specific wavelength of light. These beads are designed for use in various research and analysis applications, including flow cytometry, immunoassays, and cell tracking.
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5 protocols using yellow green fluorescent beads
1
Preparing Fluorescent Beads for iSIM
2
Antibody-Mediated Phagocytic Activity Assay
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Characterization of the phagocytic activity of serum antibodies was performed as described previously77 (link). Briefly, 1 uM yellow-green fluorescent beads (Thermo Fisher, F8813) were covalently conjugated to spike or RBD antigens. Beads were then incubated with serum samples for 4 hr with THP-1 cells (ATCC TIB-202). Cells were fixed and analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec). Scores were calculated as the percentage of cells that phagocytosed one or more fluorescent beads multiplied by the MFI of this population. S309 and VRC01 antibodies were included as positive and negative controls, respectively. Additional control wells with no added antibody were used to determine the level of antibody-independent phagocytosis. Serum samples were assayed at three different dilutions, which were determined by an initial pilot experiment to determine the optimal dilution series for measuring signal compared to background. All samples were run in three biological replicates.
3
Phagocytic Activity of SARS-CoV-2 Antibodies
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Characterization of the phagocytic activity of serum antibodies was performed80 (link). Briefly, 1 μM yellow-green fluorescent beads (Thermo Fisher, F8813) were covalently conjugated to spike or RBD antigens. Beads were then incubated with serum samples for 4 h with THP-1 cells (ATCC TIB-202) at 37 °C in 5% CO2. Cells were fixed and analyzed by flow cytometry (Supplemental Fig. 6 ) using a MACSQuant Analyzer (Miltenyi Biotec). Scores were calculated as the percentage of cells that phagocytosed one or more fluorescent beads multiplied by the MFI of this population. S309 and VRC01 antibodies were included as positive and negative controls, respectively. Additional control wells with no added antibody were used to determine the level of antibody-independent phagocytosis. Serum samples were assayed at three different dilutions, which were determined by an initial pilot experiment to determine the optimal dilution series for measuring signal compared to the background. Concentrated pooled polyclonal serum IgG (Sigma Aldrich I4506) was used as a positive control for endemic CoV (Supplemental Fig. 7 ), and samples were run in three biological replicates.
4
Intraventriculal Bead Injection in Larval Fish
5
Optimizing 2P-STED Microscope Imaging
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Imaging was performed using a custom-built upright 2P-STED microscope as previously described (see [10 (link),19 (link),23 (link)] for a complete description). In all the following, 2D-STED and z-STED refer to images acquired using pure donut or pure bottle beams, respectively. Gold bead samples were used to assess the donut or bottle beam quality, while optical resolution was assessed by imaging fluorescent beads immobilized on a glass coverslip. Samples were prepared with either gold beads (150 nm Gold nanospheres, Sigma-Aldrich) or fluorescent beads (yellow-green fluorescent beads, 170 nm in diameter, Invitrogen) placed on polylysine-coated #1 coverslips. Samples were left to dry overnight and then transferred to a microscope glass slide, where they were sealed with nail polish. In order to evaluate the effect of the coverslip tilt angle, samples were placed on a tiltable mechanical stage (TTR001/M, Thorlabs), enabling us to vary the tilt angle relative to the microscope objective axis in a precise and systematic way. STED PSFs were originally checked and optimized at a tilt angle of 0 ± 0.03°.
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