Sample solution, cell culture medium (or 1× PBS), and DI water were added to each reservoir as described in Fig. 2A . Either 10 kDa dextran conjugated Alexa Fluor®488 (Thermo Fisher Scientific) or HaloTag® Alexa Fluor®488 ligand (Promega) was used as a fluorescent sample. Two syringes (25F-GT, volume 25 μL, SGE) were connected to the microfluidic device and withdrawn by a dual syringe pump (YSP-202, YMC. Co., Ltd.). In this study, two syringes were withdrawn at the same flow rates. In order to operate the EO pump, DC voltages were applied using a programmable power supply (HVS448, Model 6000D, LabSmith).
Halotag alexa fluor 488 ligand
Manufactured by Promega
Sourced in United States
The HaloTag Alexa Fluor 488 Ligand is a fluorescent labeling agent designed for use with the HaloTag protein fusion system. It provides a covalent and specific labeling of HaloTag-fusion proteins, enabling detection and visualization of the tagged proteins in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (4 mentions)
Anti ha tag antibody for ha ins pd l1, by Cell Signaling Technology (2 mentions)
Tyramide boost kit, by Thermo Fisher Scientific (2 mentions)
Nucred dead nuclear stain, by Thermo Fisher Scientific (2 mentions)
Ti inverted, by Zeiss (2 mentions)
Halo tag ligand amine o4, by Promega (2 mentions)
Pd spintrap g 25 column, by GE Healthcare (2 mentions)
Speedvac, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Streptomycin, by Harvard Bioscience (1 mentions)
Odorants, by Merck Group (1 mentions)
Dynasore, by Bio-Techne (1 mentions)
Dulbecco s mem medium, by Harvard Bioscience (1 mentions)
Fbs superior, by Harvard Bioscience (1 mentions)
Trypsin edta solution, by Harvard Bioscience (1 mentions)
Mem non essential amino acid solution 100x, by Merck Group (1 mentions)
Paraformaldehyde pfa, by Polysciences (1 mentions)
L glutamine, by Harvard Bioscience (1 mentions)
Mowiol 4 88, by Carl Roth (1 mentions)
17 protocols using halotag alexa fluor 488 ligand
1
Microfluidic Device for Dextran and Halo Labeling
2
Visualizing CADM1 Protein Expression
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Raw 264.7 cells expressing both N-terminally Halo-tagged CADM1 variant proteins and BFP (Evrogen, Moscow, Russia) were stained with cell-impermeable HaloTag Alexa Fluor 488 Ligand (Promega) according to the manufacturers’ instructions. Fluorescence images were taken under a FLUOVIEW FV10i-DOC confocal fluorescence microscope (Olympus, Tokyo, Japan).
3
Fluorescent Immunostaining of PD-L1 Proteins
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Cells were fixed in 4% PFA for 15 min, permeabilized with 0.5% Triton X-100 for 15 min and blocked with 10% goat serum for 1hr. For frozen tumor tissues staining, 40 μm sections were pre-treated with acetone for 10 minutes. After washing with PBS, cells were stained with rat antibody against mouse PD-L1 (clone 5C5, generated in the laboratory of Dr. Gordon Freeman52 (link)), mouse antibody against human PD-L1 (clone 9A11, generated in the laboratory of Dr. Gordon Freeman50 (link)) or anti-HA tag antibody for HA-ins-PD-L1 (#2367, Cell Signaling Technology). For HA staining, cells were incubated with Tyramide Boost Kit (#B40941, Thermo Fisher Scientific) according to the manufacturer’s protocol (tyramide incubation 15 min). Then cells were incubated with NucRed Dead nuclear stain (#R37113, Thermo Fisher Scientific) for 15 min and washed with PBS. For mouse PD-L1 or human PD-L1 study, cells or tissues were stained with a donkey anti-rat or donkey anti-mouse secondary antibody (Alexa Flour 488) for 1 hr. Nuclei were stained with DAPI. For Halo-tag imaging53 (link), cells were treated with 0.5 μM HaloTag Alexa Fluor 488 Ligand (#G1002, Promega) for 15 min. Images were captured using confocal microscopy (Nikon Ti inverted or Zeiss LSM880) and assembled using Fiji software.
4
Odorant Screening in Cellular Assay
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The following chemicals were used: Dulbecco’s MEM medium (#F0435), FBS superior (#S0615), L-glutamine (#K0282), penicillin (100U/ml)/streptomycin (100U/ml) (#A2212), trypsin/EDTA solution (#L2143) (Biochrom, Berlin, Germany), MEM non-essential amino acid solution (100x) (#M7145, Sigma-Aldrich, Steinheim, Germany), Gibco® HAT supplement (#21060-017, Thermo Fisher, Dreieich, Germany),CaCl2*2H2O (#22322.295), D-glucose (#101174Y), dimethyl sulfoxide (DMSO) (#83673.230), HEPES (#441476L), potassium chloride (#26764.230), and sodium hydroxide (#28244.295) (VWR Chemicals BDH Prolabo, Leuven, Belgium), sodium chloride (#1064041000, Merck, Darmstadt, Germany), D-luciferin (beetle) monosodium salt (#E464X), HaloTag® Alexa Fluor® 488 Ligand (#G1001, Promega, Madison, USA), Dynasore (#2897, Tocris Bioscience, Bristol, UK), Hoechst33342 (#1399, Invitrogen, Eugene, USA), Mowiol 4-88 (#0713, Carl Roth GmbH + Co. KG, Karlsruhe, Germany), Paraformaldehyde (PFA) (#18814, Polysciences Inc., Warrington, USA).
Odorants were purchased from Sigma-Aldrich (Steinheim, Germany), Alfa-Aesar (Karlsruhe, Germany) and Chemos GmbH (Regenstauf, Germany) (Table S1
Odorants were purchased from Sigma-Aldrich (Steinheim, Germany), Alfa-Aesar (Karlsruhe, Germany) and Chemos GmbH (Regenstauf, Germany) (
5
Monitoring Autophagy Flux Using HaloTag-LC3
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HaloTag-LC3 assays were performed as previously described (Takahashi et al., 2018 (link)). Briefly, cells were transfected with HaloTag-LC3 for 24 h and treated with 20 μM digitonin in KHM buffer at 37°C for 2 min. Then cells were incubated with membrane-impermeable ligand (MIL) HaloTag® Alexa Fluor® 488 ligand (G1001, Promega) at 37°C for 15 min. Cells were fixed in 4% PFA for 20 min at room temperature, washed 3 times with PBS, and stained with membrane-permeable ligand (MPL) HaloTag® TMR ligand (G8251, Promega) for 30 min at room temperature. Coverslips were mounted on microscope slides with DAPI in 50% glycerol and examined by a confocal microscope (LSM 880 Meta plus Zeiss Axiovert zoom, Zeiss).
6
Fluorescent Immunostaining of PD-L1 Proteins
7
HaloTag and Antibody Labeling for Flow Cytometry
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293FT cells (1×105) were transfected with the construct of interest in triplicate. At 48 h post-transfection, HaloTag Alexa Fluor 488 Ligand (Promega) was used to label HaloTag as described above. After washing with phosphate-buffered saline (PBS), cells were detached and resuspended in PBS with 3 mM EDTA, followed by 4% paraformaldehyde fixation; alternatively, 1 µg/mL V3-G2-25 antibody [25] (link) was added to the cells after PBS washing and incubated at 4°C for 30 min. Then washed cells were incubated at 4°C for 30 min with 1 µg/mL Alexa Fluor 488 donkey anti-mouse IgG antibody (Invitrogen). Finally, the cells were resuspended in PBS with 5% FBS and 4% paraformaldehyde. Flow cytometry data were acquired with a BD FACSCalibur system (Becton, Dickinson and Company), at least 10,000 events were collected and analyzed using FlowJo software (Tree Star).
8
BMPR2 Localization in HMEC-1 Cells
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HMEC‐1‐HaloTag‐BMPR2 cells were treated overnight with BafA1 (20 nm ). Next, they were incubated in MCDB131 media containing the non‐permeable HaloTag Alexa Fluor 488 ligand (G1001; Promega) at a dilution of 1:10 × 103 for 15 min at 37°C. Cells were then washed three times with medium and images were recorded on a Leica DMi1 microscope.
9
Halo-JF-646 Fluorescent Protein Labeling
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Fluorescent dye JF-646 (Grimm et al., 2015 (link)) NHS-ester building block (TOCRIS) was conjugated with Halo-tag ligand amine O4 (Promega) by synthetic chemistry according to published protocols (Grimm et al., 2017 (link)). Briefly, 1.5 equivalents of amine O4 ligand were added to one equivalent of the JF-646 NHS-ester in DMF followed by adding 5% triethylamine. The reaction was vigorously stirred for 16 hr at room temperature and the product was purified by silica gel chromatography, dried by SpeedVac (ThermoFisher), and reconstituted in DMSO.
For optical trapping/confocal microscopy assays, HaloTag Alexa Fluor 488 Ligand (Promega) was utilized as described above, followed by desalting through a PD SpinTrap G-25 column (GE Healthcare) according to the manufacturer’s protocol to remove unreacted dye before use. To label the Halo-tagged actin-binding proteins with Halo-JF-646 for TIRF microscopy assays, two equivalents of synthesized Halo-JF-646 dye was added to the protein solution, followed by incubation for at least 2 hr in the dark at 4°C before use. Subsequent removal of excess dye was not required, as JF-646 is a fluorogenic dye (Grimm et al., 2015 (link)).
For optical trapping/confocal microscopy assays, HaloTag Alexa Fluor 488 Ligand (Promega) was utilized as described above, followed by desalting through a PD SpinTrap G-25 column (GE Healthcare) according to the manufacturer’s protocol to remove unreacted dye before use. To label the Halo-tagged actin-binding proteins with Halo-JF-646 for TIRF microscopy assays, two equivalents of synthesized Halo-JF-646 dye was added to the protein solution, followed by incubation for at least 2 hr in the dark at 4°C before use. Subsequent removal of excess dye was not required, as JF-646 is a fluorogenic dye (Grimm et al., 2015 (link)).
10
Visualizing CRT-HaloTag-KDEL Localization
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For CRT analysis, Turbofectin 8.0 (Origene) was used to transfect TSA cells with a pEZ-M02 expression vector containing the Mus musculus CRT-HaloTag-KDEL fusion construct (refer to Fig. S1 ) following the manufacturer’s instructions (GeneCopoeia). Stable clones were selected by means of G418 selection (Gibco). To validate and detect CRT-HaloTag-KDEL endoplasmic reticulum localization, cloned cells were incubated overnight with 5 µM R110DirectTM ligand (Promega), diluted in phenol free medium, according to the manufacturers instructions. CRT-HaloTag-KDEL bound R110DirectTM ligand, a small molecule that crosses the cell membrane and labels total HaloTag® fusion protein, was visualized on an EVOS fluorescence microscope (Advanced Microscopy Group) at exλ470 nm/emλ525 nm. Positive clones were chosen for subsequent experiments. To visualize CRT-HaloTag-KDEL membranous localization, cloned cells were incubated with the 5 µM of the cell membrane impermeable HaloTag® Alexa Fluor 488 ligand (Promega) for 30 min and processed according to the manufacturers instructions. CRT-HaloTag-KDEL bound HaloTag® Alexa Fluor 488 ligand was visualized on an EVOS fluorescence microscope (Advanced Microscopy Group) at exλ470 nm/emλ525 nm.
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