HEK293 cells were seeded to PDL-coated coverslips (Neuvitro #GG12PDL) such that they reached 50% confluency on day of transfection. Cells were transfected with 500 ng of pTX066 or pEGFP-C1 using Fugene 6 transfection reagent per manufacturer protocol. Forty-eight hours post-transfection, cells were fixed in 4% paraformaldehyde (EMS, Hatfield PA) for 10 minutes at room temperature followed by washing in 1X PBS and permeabilization in 0.2% Triton X-100/PBS for 8 minutes. Cells were rinsed in PBS and blocked in 2.0% non-fat milk made up in 0.2% TBS-T for 1 hour at room temperature. Cells were incubated with HSPA1A/B C92F3A-5 (1:500) in blocking buffer for 16 hours at 4°C. Cells were washed 3X in TBS-T and incubated with Alexa-fluor 647-conjugated anti-mouse secondary antibody (Invitrogen # A-28181, 1:500) for 1 hour at room temperature and counterstained with 100 nM DAPI in PBS. Coverslips were mounted with ProLong Diamond Antifade mountanti (Invitrogen, #P36961).
Alexa fluor 647 conjugated anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa-Fluor 647-conjugated anti-mouse secondary antibody is a fluorescently labeled antibody used to detect and visualize mouse primary antibodies in various immunoassays and imaging applications. The Alexa-Fluor 647 dye provides bright, photostable fluorescence for sensitive detection.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Rabbit anti lrp6, by Santa Cruz Biotechnology (1 mentions)
Mouse anti active β catenin, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Harmony software, by PerkinElmer (1 mentions)
F12 media, by Thermo Fisher Scientific (1 mentions)
Operetta high content imaging system, by PerkinElmer (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Operetta cls high content imaging system, by PerkinElmer (1 mentions)
Mccoy s 5a media, by Thermo Fisher Scientific (1 mentions)
Versene, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Anti c myc monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Facscanto 2, by BD (1 mentions)
6 protocols using alexa fluor 647 conjugated anti mouse secondary antibody
1
Immunofluorescence Staining of HEK293 Cells
2
Fluorescent Lipid Raft and Protein Localization
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Before fixation, lipid rafts were labeled with Vybrant lipid rafts labeling kit (Invitrogen). Cells cultured on coverslips were incubated with 0.5mM fluorescent Cholera Toxin B-Subunit (CT-B, Alexa 594) for 10 minutes at 4°C. After washing with PBS, cells were treated with anti-CT-B antibody (dilution 1:200) for another 10 minutes at 4°C. In the following fixation and immunofluorescence staining was performed as described previously [70 (link)]. Accordingly, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min (Sigma-Aldrich). To reduce non-specific binding, cells were treated with 1% gelatin. First, cells were labeled with rabbit anti-LRP6 (Santa Cruz, dilution 1:150) and subsequently incubated with Alexa Fluor 488 (Invitrogen, dilution 1:300). Afterwards, cell membranes were permeabilised with 0.2% Triton X-100 (Sigma-Aldrich) followed by labelling with mouse anti-active-β-catenin (Millipore, dilution 1:250) and subsequent incubation with Alexa Fluor 647-conjugated anti-mouse secondary antibody (Invitrogen, dilution 1:300) and Hoechst for nuclei staining (Sigma-Aldrich, dilution 1:1000). Finally, cells were mounted on microscope slides using ProLong Gold antifade reagent (Invitrogen).
3
Quantifying GPCR Internalization in Cells
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Example 8
Internalization Assay
GLP-1R and GIPR internalization were assessed in CHOK1 cells expressing both human GLP-1R and GIPR. Cells were plated at a density of 25,000 cells/well in 96 well plates and cultured overnight at 37° C., 5% CO2. Cells were serum starved in F12 media (Thermo Fisher) with 0.1% BSA for 4 hours prior to treatment. Cells were treated with 20 nM or dose titrations of GLP-1, GIP or bispecific conjugates for 30 minutes or the specified time in the respective figures. Cells were washed and fixed with 4% formaldehyde and permeabilized with 0.1% triton-X 100. To detect GIPR or GLP-1R, cells were first blocked with Odyssey blocking buffer (LiCor) for 1 hour at room temperature and incubated with 2 μg/ml of mouse anti-human GLP-1R (R&D systems) or 5 μg/ml of anti-human GIPR antibody (R&D Systems) at 4° C. overnight followed by AlexaFluor-555 or Alexa-Fluor 647 conjugated anti-mouse secondary antibody (Thermo Fisher) for GLP-1R or GIPR detection, respectively. Images (
4
Co-Localization of GLP-1R, GIPR, and Bispecific Conjugate
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Example 9
GLP-1R, GIPR and Bispecific Conjugate are Co-Localized Upon Internalization
U2OS cells expressing GIPR and SNAP-GLP-1R were plated at a density of 15,000 cells per well in 96 well plate and cultured overnight at 37° C. with 5% CO2. Prior to treatment, cells were starved for 3.5 hours in McCoy's 5A media (Thermo Fisher) with 0.1% BSA. Prior to treatment, GLP-1R on the surface of U2OS cells were labeled with Alexa-Fluor 564 by incubating with SNAP-Surface Alexa-fluor 564 substrate (New England Biolabs) for 30 minutes. Cells were then washed to remove excess label and treated with 5 nM bispecific conjugates for 30 minutes. After 3 washes, cells were fixed with 4% formaldehyde and permeabilized with 0.1% triton-X 100 in PBS. For GIPR detection, cells were first blocked with Odyssey blocking buffer (LiCor) for 1 hour at room temperature and then incubated with 5 μg/ml of mouse anti-human GIPR (R&D Systems) at 4° C. overnight followed by incubation with Alexa-Fluor 647 conjugated anti-mouse secondary antibody (Thermo Fisher) for 1 hour at room temperature. Bispecific conjugate was detected using Alexa-Fluor labeled anti-Human Fc antibody. Hoechst 33342 were used for nuclei detection (Thermo Fisher). Images were captured using Operetta CLS high content Imaging system (Perkin Elmer) as shown in
5
Flow Cytometry Analysis of CD44v6 Antibody Binding
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Binding affinity of CD44v6 half-antibody fragments to GC cells was analyzed by flow cytometry. MKN74 and CD44v6 cells cultured in T75 flasks were harvested with Versene (Gibco, Paisley, UK) and blocked with flow cytometry buffer containing 10% FBS and 0.1% (v/v) sodium azide in PBS for 30 min on ice. Afterward, 2 × 105 cells were incubated with either CD44v6 antibody (1:200) or CD44v6 half-antibody fragments (TCEP 5–1000 μM) (1:100) in flow cytometry buffer for 1 h on ice followed by three washing steps. After 1 h of incubation with anti-mouse Alexa Fluor 647-conjugated secondary antibody (Thermo Scientific, Rockford, IL, USA), cells were fixed with 4% (v/v) paraformaldehyde (PFA) for 20 min at RT and finally resuspended in PBS. Cell-associated fluorescence was measured with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed with FlowJo v8.7.
6
CRHR1 Surface Expression in HT22 Cells
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For CRHR1 surface staining, HT22-CRHR1 cells on 60-mm plates were starved for 1 h in OptiMEM before drug pretreatments or stimulation. When pharmacological inhibitors were used, cells were pretreated with drugs or vehicle 15–30 min before stimulation with CRH for the indicated concentration and time. After incubation, cells were washed with ice-cold PBS and harvested in 1 mM PBS-EDTA. 2.0 × 105 cells were incubated at 4 C with 10% FBS in PBS for 1 h to reduce nonspecific binding. Subsequently, cells were incubated with 0.2 µg anti–c-Myc monoclonal antibody (Santa Cruz Biotechnology, Inc.) for 2 h and, after washing with 1% FBS in PBS, were stained with 1.4 µg anti-mouse Alexa Fluor 647–conjugated secondary antibody (Thermo Fisher Scientific). All solutions contained 0.01% sodium azide. Controls for autofluorescence, primary antibody staining on parental HT22 cells, and secondary antibody staining in absence of primary antibody were performed for each experiment. Flow cytometry data were acquired on a FACsCANTO II (BD). Data were analyzed using FlowJo software (Tree Star).
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