HeLa cells stably expressing Escherichia coli glutamine synthetase GlnA-HaloTag (Finan et al., 2015 ) were a gift of Florian Salopiata (DKFZ Heidelberg). U2OS cells stably expressing NUP107-SNAP-tag were a gift of Jan Ellenberg (EMBL Heidelberg) (Otsuka and Ellenberg, 2017 (link)). Both cell lines were labeled with corresponding tag substrates directly before fixation. GlnA-HaloTag–expressing cells were labeled with TMR HaloTag ligand (HTL-TMR; Promega, USA) or SiR HaloTag ligand (SiR-HTL; Spirochrome, Switzerland) at 100 nM in growth medium for 120 min at 37°C. NUP107-SNAP-tag–expressing cells were labeled with benzylguanine-functionalized TMR (BG-TMR; NEB, USA) or SiR (BG-SiR; Spirochrome) at 200 nM in growth medium for 120 min at 37°C. After labeling, cells were washed repeatedly with growth medium and fixed as described above.
Htl tmr
Manufactured by Promega
Sourced in United States
The HTL-TMR; is a laboratory instrument designed for high-throughput liquid handling tasks. It features automated pipetting, sample mixing, and temperature control functionalities to support various laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using htl tmr
1
Labeling HeLa and U2OS cell lines
2
Tracking of Single Molecules in Cells
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Localization and tracking of single molecules were done as previously described16 (link). Briefly, infected HeLa LAMP1-GFP or RAW264.7 cells were labeled with the HaloTag ligand coupled to tetramethylrhodamine (HTL-TMR, Promega G8251) in a concentration of 20 nM for 15 min at 37 °C. After 10 washing steps, the cells were imaged with an imaging medium consisting of Minimal Essential Medium (MEM) with Earle’s salts, without NaHCO3, without L-Glutamine, without phenol red (Merck D1145) and supplemented with 30 mM HEPES, pH 7.4. TIRF microscopy was performed using an inverted microscope Olympus IX-81, equipped with an incubation chamber maintaining 37 °C and humidity, a motorized 4-line TIRF condenser, a ×150 objective (UAPON 150x TIRF, NA 1.45), a TIRF quadband polychroic mirror (zt405/488/561/640rpc), a 488 nm laser (150 mW, Olympus), and a 561 nm laser (150 mW, Olympus). Localization, as well as tracking of single molecules, were carried out with the help of a self-written user interface in MatLab 2013a21 ,46 (link)–50 (link).
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