CHO-K1 cells were transfected with pFLAG-TREK1 for 24 h. After washed with phosphate-buffered saline (PBS), the cells were disassociated with a non-enzyme cell dissociation solution (Sigma). After 10 μM TKDC incubation for 20, 40 min and 1 h at room temperature, cell surface TREK-1 expression in CHO-K1 cells was analyzed by flow cytometry. Transfected cells expressing FLAG-tagged TREK-1 were incubated with monoclonal ANTI-FLAG® M2-FITC antibody (Sigma). Data were collected with a flow cytometer (FACS Calibur, BD Bioscience) and analyzed with Flow Jo.
Anti flag m2 fitc antibody
Manufactured by Merck Group
The Anti-FLAG M2-FITC antibody is a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody that specifically binds to the FLAG peptide tag. The FLAG tag is a widely used protein tag that allows for the detection and purification of recombinant proteins. The Anti-FLAG M2-FITC antibody can be used to detect and visualize FLAG-tagged proteins in various applications such as Western blotting, immunoprecipitation, and immunocytochemistry.
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Lab products found in correlation
Envision multimode plate reader, by PerkinElmer (2 mentions)
Facscalibur, by BD (1 mentions)
Goat serum, by Cell Signaling Technology (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi media, by Thermo Fisher Scientific (1 mentions)
Image it fx signal enhancer, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Formalin, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Glosensor camp assay kit, by Promega (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Glosensor camp assay, by Promega (1 mentions)
Prism 7, by GraphPad (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
8 protocols using anti flag m2 fitc antibody
1
TREK-1 Surface Expression in CHO-K1 Cells
2
Immunostaining of Transfected A375 Cells
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A375 cells were maintained in RPMI media (Invitrogen) supplemented with 5% (v/v) FBS (Invitrogen), 100 U ml−1 penicillin (Invitrogen), and 100 g ml−1 streptomycin (Invitrogen). Cells were cultured in a humidified 5% CO2 incubator at 37 °C. 25,000 cells were placed on poly-l -lysine-coated coverslips seeded in 24-well plates and incubated to adhere for 24 h. pcDNA-ERK2-WT or mutant was transfected using Lipofectamine™ 3000 (Invitrogen) reagent according to the manufacturer’s protocol. Cells were incubated for another 36 h, then maintained for one more night in serum-free media. Cells were treated with 25 µM of BI-78D3 for 2 h and induced with EGF for 15 min, followed by washing using PBS and fixation in 4% paraformaldehyde in PBS for 10 min at room temperature. The cells were permeabilized with 0.2% Triton X-100 for 5 min and blocked with 10% Goat serum (Cell Signaling Technology). Image-iT™ FX signal enhancer (Invitrogen) was used according to the manufacturer’s protocol. Immunostaining was performed by incubating cells with monoclonal ANTI-FLAG® M2-FITC antibody produced in mouse (F4049-Sigma). DAPI (4′, 6′-diamidino-2-phenylindole) was included in the mounting medium as a counterstain for nuclei and was visualized by the A Zeiss Axiovert 200M microscope with ×63 magnification.
3
FAP and CD3 Binding Assay
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Binding assays were performed with HT1080 cells transfected with either human or murine FAP antigen or CD3+ Jurkat cells. HT1080 (2 × 105) or Jurkat (1 × 105) cells were incubated on ice for one hour with the concentrated or unconcentrated supernatants. BiTE binding was determined by flow cytometry using anti-FLAG M2-FITC antibody (Sigma Aldrich).
4
Subcellular Localization of RORα
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1x106 splenocytes were treated with PBS, 2 μM nt-RORα-TMD, or 2 μM RORα-TMD in 96 well-plate and incubated. The splenocytes were harvested and seeded on top of a round microscope cover glass 18-mm in diameter placed on the bottom of a 12-well culture plate. Cells were fixed with 10% formalin (Sigma-Aldrich) and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich). After blocking with 1% BSA in PBS, anti-FLAG M2-FITC antibody (Sigma-Aldrich) was used to stain the recombinant proteins. 4ʹ,6-diamino-2-phenylindole (DAPI) was used to counterstain nuclei, and cells were analyzed with a confocal microscope (Carl Zeiss).
5
Measuring Basal Activity of mGlu2-mGlu3 Heterodimers
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To measure the basal activity of mGlu2–mGlu3, IP accumulation assay was performed by using IP-One Gq assay kit (PerkinElmer) following the manufacturer’s instructions. The wild-type and mutant mGlu2 and mGlu3 genes, which contain the GABAB tails, were cloned into the pTT5 vector. The HEK293F cells were co-transfected with the plasmids of mGlu2, mGlu3, the chimeric Gα protein Gαqi959 (link) and EAAT1 at a ratio of 1:1:2:1 with the final concentration of 1 μg/mL. The heterodimers were expressed following the same protocol of the BRET assay. After 48 h, surface expression levels were measured with the monoclonal anti-Flag M2-FITC antibody (Sigma; 1:100 diluted in TBS + 4% BSA) using a flow cytometry reader (Millipore). The cells were then loaded into 384-well white plates (40,000 cells per well) and incubated with stimulation buffer (provided by manufacturer) at 37 °C for 90 min. The basal activity of mGlu2–mGlu3 was calculated by subtracting the IP production measured in the control (cells co-transfected with Gαqi9, EAAT1, and the empty pTT5 vector) for the wild-type heterodimer and all the mutants.
6
GLP-1R Expression and cAMP Signaling
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HEK-293T
cells were plated in 6-well dishes at a density of 500 000–600 000
cells per well, cultured in DMEM supplemented with 10% (v/v) fetal
bovine serum, and maintained in the incubator at 37 °C and 5%
CO2. After overnight of culture, specified amounts of wild-type
or mutant GLP-1R and 0.5 μg of pGloSensor cAMP plasmid were
cotransfected into HEK-293T cells using calcium phosphate. After 24
h of culture, the transfected cells were seeded into 384-well plates
(12 000 cells per well). cAMP accumulation was measured using
the GloSensor cAMP assay kit (Promega) according to the manufacturer’s
instructions. In brief, transfected cells were incubated for 30 min
in GloSensor cAMP reagent at 37 °C and room temperature. Wild-type
or mutant GLP-1R were stimulated with different concentrations of
GLP-1 for 15 min at 25 °C and were measured by an EnVision multimode
plate reader (PerkinElmer).
To evaluate the cell surface expression
of wild-type and mutant GLP-1R, the remaining cells after seeding
into 384-well plates were incubated with anti-FLAG M2-FITC antibody
(Sigma) or anti-HA–FITC antibody (Sigma) for 30 min at 4 °C
and were measured by Beckman Coulter CytoFLEX flow cytometer.
cells were plated in 6-well dishes at a density of 500 000–600 000
cells per well, cultured in DMEM supplemented with 10% (v/v) fetal
bovine serum, and maintained in the incubator at 37 °C and 5%
CO2. After overnight of culture, specified amounts of wild-type
or mutant GLP-1R and 0.5 μg of pGloSensor cAMP plasmid were
cotransfected into HEK-293T cells using calcium phosphate. After 24
h of culture, the transfected cells were seeded into 384-well plates
(12 000 cells per well). cAMP accumulation was measured using
the GloSensor cAMP assay kit (Promega) according to the manufacturer’s
instructions. In brief, transfected cells were incubated for 30 min
in GloSensor cAMP reagent at 37 °C and room temperature. Wild-type
or mutant GLP-1R were stimulated with different concentrations of
GLP-1 for 15 min at 25 °C and were measured by an EnVision multimode
plate reader (PerkinElmer).
To evaluate the cell surface expression
of wild-type and mutant GLP-1R, the remaining cells after seeding
into 384-well plates were incubated with anti-FLAG M2-FITC antibody
(Sigma) or anti-HA–FITC antibody (Sigma) for 30 min at 4 °C
and were measured by Beckman Coulter CytoFLEX flow cytometer.
7
Evaluating Mutant EP2 Receptor Activity
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The coding sequence of WT human EP2 followed by a hemagglutinin signal peptide and Flag tag at the N terminus was subcloned into the pcDNA3.1 vector. Point mutations of EP2 were generated using the Q5 site-Directed Mutagenesis kit (NEB). The WT and mutant EP2 constructs were transfected into COS-7 cells and harvested at 48 hours after transfection. Expression level of the WT and mutant EP2 receptors was determined by flow cytometry with an anti-Flag M2-FITC antibody (Sigma-Aldrich, F4049). cAMP level was measured using GloSensor cAMP assay (Promega) for EP2 as previously described (34 (link)). For GloSensor cAMP assay, WT and mutant EP2 and the Glosensor plasmid were cotransfected into COS-7 cells. The infected cells were plated into 96-well plates and cultured for 48 hours at 37°C with 5% CO2. A serum-free medium with 5% v/v dilution of the GloSensor cAMP reagent stock solution (Progema) was used to incubate with the EP2 plasmid transfected cells. Afterward, a range of concentrations of the EP2 ligands were added, and the luminescence signals were counted on an EnVision multimode plate reader (PerkinElmer). cAMP level was calculated according to a standard dose-response curve. EC50 (half maximal effective concentration) was calculated via nonlinear regression (curve fit) using GraphPad Prism 7 (GraphPad Software). Each measurement was performed in triplicate.
8
Quantifying PAR4 Cleavage in COS7 Cells
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