Purified TFG-mEGFP-FLAG (+truncations) in 50 mM HEPES/KOH, pH 7.3 and 150 mM KCl was pipetted onto standard glass-bottom MatTek dishes (P35GC-1.5–14-C) to form condensates (with 20% v/v PEG 8 kDa as needed) and overlaid with fluorescent dextrans of various sizes at 10 kDa (Invitrogen D1816), 40 kDa (Invitrogen D1842), 70 kDa (Invitrogen D1819), 155 kDa (T1287–100MG, SIGMA-ALDRICH, INC.), 250 kDa (TMR-dex 250 kDa, Fina Biosolutions), and 500 kDa (52194–1G, SIGMA-ALDRICH, INC.) suspended in 50 mM HEPES/KOH, pH 7.3 and 150 mM KCl. Dextran species were added at a final concentration of 1 µM and incubated for 10 minutes. All images were taken in confocal mode using the OMX Flex SR microscope.
D1816
Manufactured by Thermo Fisher Scientific
The D1816 is a laboratory centrifuge designed for general-purpose applications. It features a maximum speed of 4,000 RPM and a maximum relative centrifugal force (RCF) of 2,000 x g. The centrifuge can accommodate a variety of rotor types and sample volumes.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using d1816
1
Condensate Formation and Dextran Partitioning
2
Dextran-based Molecular Thermometry for High-speed Imaging
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Water soluble tetramethylrhodamine attached to 10,000 MW dextran (TMR-Dextran) acts as the molecular thermometer. The fluorophore solution was prepared by dissolving the TMR-Dextran powder (Invitrogen, D1816) in distilled water (10 mg/ml). Even at the fastest recording times of 90,000 fps, the exposure time is much longer than the relaxation time of TMR which is on the order of nanoseconds.
Figure 5 shows the relative fluorescent intensity recorded at 54,000 fps for more than 0.35 s (bold line, upper timescale), which greatly exceeds the duration of each experimental run. Hence we can discard photobleaching due to the excitation lamp. Also in Fig. 5 we show some of the data for the calibration (100 frames, lower timescale).
Supplementary Video 1 . Video for the case shown in Figure 2 (evolution of the temperature distribution), 102 × 102 µm (W × H) filtered with a circular averaging filter with a radius of four pixels. The total duration is 6.5 milliseconds.
3
Dextran Uptake Quantification in Mouse ACC
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Cultured mouse ACCs were washed 3 times with cold standard extracellular bath solution, then incubated for 2 min with 50 µM dextran (10 and 40 kDa, Invitrogen, D1816, and D1842, respectively) in 37 °C 70 mM KCl containing external solution. Unbound dye was washed out with standard cold 0 Ca2+ immediately after incubation. The cells were then incubated in cold 0 Ca2+ solution. Z‐series of 1‐µm optical sections were scanned through the 40×oil‐immersion lens of a Zeiss 710 inverted confocal microscope. The consecutive optical sections were z‐projected and the total numbers of dextran fluorescent puncta per cell were determined. Images were processed with ImageJ (NIH).
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