The 2D distances between MCP-tagged RNA and SNAP-tagged RF clusters or dCas9-Halo-tagged genomic loci were estimated from the coordinates of the fitted 2D Gaussians in the respective images. The two colors were aligned and overlapped first by measuring the back-scattered light of the 490nm and 642nm laser excitation beams from ~100nm Au nanoparticles, and then by imaging 40nm-diameter TransFluoSpheres (488/645; ThermoFisher Scientific, T10711). Residual systematic offsets (typically < 30nm) were then subtracted for the final 2D distance measurements. The effect of chromatic aberrations for 2D distance measurements in live cells was further estimated by measuring transcription sites with doubly-tagged nascent RNA. Specifically, we tagged the 24×PP7 nascent RNA of a mini-gene, driven by the CMV promoter in U-2 OS cells39 (link), simultaneously with tdPCP-EGFP and tdPCP-Halo-JF646. From this type of measurements, we estimate ≤29 nm r.m.s. registration errors between EGFP or mNeonGreen and SiR or JF646, well below the range of typical genomic distances measured (~100–300 nm).
Transfluospheres
Manufactured by Thermo Fisher Scientific
TransFluoSpheres are fluorescent microspheres designed for use in a variety of life science applications. They are available in multiple colors and sizes to suit different experimental needs.
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7 protocols using transfluospheres
1
Measuring Genomic Loci Distances
2
Frustrated Phagocytosis Microscopy Protocol
3
Frustrated Phagocytosis Microscopy Protocol
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Glass coverslips #1.5 were coated with neutravidin (Thermo scientific) at 0.5 mg/mL in PBS, at 4°C for 4 hours. After washes, coverslips were incubated with 10 μg/mL biotin-conjugated M1/70 (BD Pharmingen 553309) or rat IgG2b isotype control (BD Pharmingen 553987) antibodies at 4°C overnight. Coverslips were then blocked with PLL-PEG (Susos) at 0.1 mg/mL, at 4°C for at least 2 hours. Macrophages were suspended in ice cold PBS with 2 mM EDTA by scraping, then washed in serum free RMPI 1640, supplemented with 25 mM HEPES pH 7.3 and 150 ng/ml PMA 15 minutes before addition onto the coverslips. Cells were imaged immediately for live cell microscopy, or fixed with 4% PFA after 5 minutes and processed for immunofluorescence.
For frustrated phagocytosis on polyacrylamide gels and traction force microscopy, 40 μm thick gels with 40 nm TransFluoSpheres (633/720) (Thermo scientific) were prepared on glass coverslips as described before35 (link). neutravidin (0.5 mg/mL) was crosslinked to the surface of the gels using Sulfo-SANPAH at 1mg/ml (Thermo scientific). After washes, gels were incubated with biotin-conjugated M1/70 (BD Pharmingen 553309) or rat IgG2b isotype control (BD Pharmingen 553987) antibodies at 10 μg/mL, at 4°C overnight.
For frustrated phagocytosis on polyacrylamide gels and traction force microscopy, 40 μm thick gels with 40 nm TransFluoSpheres (633/720) (Thermo scientific) were prepared on glass coverslips as described before35 (link). neutravidin (0.5 mg/mL) was crosslinked to the surface of the gels using Sulfo-SANPAH at 1mg/ml (Thermo scientific). After washes, gels were incubated with biotin-conjugated M1/70 (BD Pharmingen 553309) or rat IgG2b isotype control (BD Pharmingen 553987) antibodies at 10 μg/mL, at 4°C overnight.
4
Measuring B Cell Contractile Forces
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Polyacrylamide gels (PA, 0.23 kPa shear modulus, 40 μm thickness) were prepared on glass coverslips with embedded 40 nm fluorescent beads (TransFluoSpheres [633/720], Thermo Scientific), as described previously (Jaumouillé et al., 2019 (link)). B cells were resuspended in mHBS with 2% FCS and added to PA gels. Images of B cells that had engaged PA gels for 20 min were captured. A no-stress reference image of the PA gels with beads was captured after lifting cells from the PA gel by adding 1% sodium dodecyl sulfate in 1× PBS to the imaging chamber at a final concentration of 0.04%. Particle image velocimetry was used to calculate bead displacements relative to the reference position, and the corresponding contractile energy was quantified using ImageJ plugins as previously described (Jaumouillé et al., 2019 (link); Martiel et al., 2015 (link)). Traction forces were reported as the mean magnitude of traction stress within the cell relative to the cell surface area.
5
Measuring Genomic Loci Distances
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The 2D distances between MCP-tagged RNA and SNAP-tagged RF clusters or dCas9-Halo-tagged genomic loci were estimated from the coordinates of the fitted 2D Gaussians in the respective images. The two colors were aligned and overlapped first by measuring the back-scattered light of the 490nm and 642nm laser excitation beams from ~100nm Au nanoparticles, and then by imaging 40nm-diameter TransFluoSpheres (488/645; ThermoFisher Scientific, T10711). Residual systematic offsets (typically < 30nm) were then subtracted for the final 2D distance measurements. The effect of chromatic aberrations for 2D distance measurements in live cells was further estimated by measuring transcription sites with doubly-tagged nascent RNA. Specifically, we tagged the 24×PP7 nascent RNA of a mini-gene, driven by the CMV promoter in U-2 OS cells39 (link), simultaneously with tdPCP-EGFP and tdPCP-Halo-JF646. From this type of measurements, we estimate ≤29 nm r.m.s. registration errors between EGFP or mNeonGreen and SiR or JF646, well below the range of typical genomic distances measured (~100–300 nm).
6
Traction Force Measurements during Macrophage Phagocytosis
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Polyacrylamide gels (4.07 kPa shear modulus, 40 um thickness) were prepared on glass coverslips with embedded 40 nm fluorescent beads (TransFluoSpheres (633/720), Thermo scientific) as described before35 (link). An average of 10 images of the beads in the gels was acquired before addition of macrophages as a no-stress reference. Macrophages were added and beads (as markers of gel deformation deformation) and expressed fluorescent proteins imaged every 10 seconds during frustrated phagocytosis. Bead displacements relative to the reference position were determined using particle image velocimetry, and the corresponding contractile energy was estimated with the Fourier transform traction cytometry, using the ImageJ plugins previously described61 (link). The phagocytic cup area was determined by manually drawing a region of interest following the leading edge of the cup in the fluorescent protein channel. Maximal and mean stress within the phagocytic cup were measured at the time point when the cup reached its maximal surface area. For measurement of traction force over time, mean magnitude of traction stress within the cup was measured and reported to the surface area of the cup.
7
Traction Force Measurements during Macrophage Phagocytosis
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