SH-SY5Y or SW2 cells were plated at 20,000 cells/cm2 on poly-L-lysine coated 35 mm glass bottom dishes and adhered overnight. To remove polySia, cells were treated as described above. To measure receptor-antibody internalization, cells were incubated with 150 nM AF488 labeled ch735, human IgG isotype control (ThermoFisher Scientific), mo735, or mouse IgG isotype control (anti-MBP mAb, NEB) and 100 nM AF647 transferrin or AF647 anti-LAMP-3 antibody (Santa Cruz) for 1 h. Cells were washed and then fixed as described above. To examine lysosomal trafficking, cells were incubated with 150 nM of ch735 or isotype at 37°C for 2 h, washed, fixed, and then permeabilized using 0.1% Triton X-100 NPBS. Cells were stained with AF488-labeled antibody against human IgG to visualize antigen-antibody complex (ThermoFisher Scientific) and mouse anti-human LAMP-1 clone D2D11 (Cell Signaling) followed by AF647-labeled anti-rabbit IgG to visualize the lysosomes (ThermoFisher Scientific). Hoescht dye was used at 1 μg/mL in PBS for 5 min at room temperature. Samples were imaged on a Zeiss LSM inverted 880 confocal microscope using a 40x water immersion objective. For colocalization analysis, a 5-μM line was drawn across the apparent vesicles. The fluorescence intensity of the plot profile was analyzed using FIJI software. Fluorescence intensity was normalized to the maximum value for each channel.
Human igg isotype control
Manufactured by Thermo Fisher Scientific
The Human IgG isotype control is a laboratory reagent used in immunoassays to establish background signal levels. It serves as a negative control to determine non-specific binding of antibodies in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ab72469, by Abcam (2 mentions)
Ab72569, by Abcam (2 mentions)
Lsm 880 inverted confocal microscope, by Zeiss (1 mentions)
Ctla4 ig, by Bristol-Myers Squibb (1 mentions)
Freund s complete adjuvant, by Fujifilm (1 mentions)
Nk cell enrichment kit, by STEMCELL (1 mentions)
Chromium 51 51cr, by PerkinElmer (1 mentions)
Wizard automatic gamma counter, by PerkinElmer (1 mentions)
Aducanumab, by Cardinal Health (1 mentions)
Novaseq s2, by Illumina (1 mentions)
6 protocols using human igg isotype control
1
Quantifying Receptor-Antibody Internalization
2
HEK293FT Cell Surface Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For surface staining, HEK293FT cells were plated at 3 × 105 cells per well in 1 ml DMEM in 12-well plates and transfected as described above, using 200 μl transfection reagent per well. At 36–48 h after transfection, cells were harvested with 500 µl FACS buffer and spun at 150×g at 4°C for 5 min. For the experiment in which multiple harvest methods were compared (Supplementary Figure S14 ), some samples were harvested using 0.05% Trypsin-EDTA (3 min or 10 min incubation, 37°C), which were then quenched with medium and added to two volumes of FACS buffer. Supernatant was decanted, and 50 µl fresh FACS buffer and 10 µl human IgG (Human IgG Isotype Control, ThermoFisher Scientific #02-7102, RRID: AB_2532958, stock concentration 1 mg/ml) was added. Cells were incubated in this mixture at 4°C for 5 min. Next, 5 µl FLAG tag antibody (Anti-DDDDK-PE, Abcam ab72469, RRID: AB_1268475 or Anti-DDDDK-APC, Abcam ab72569, RRID: AB_1310127) was added at a concentration of 0.5 µg per sample and cells incubated at 4°C for 30 min. Following incubation, 1 ml FACS buffer was added, cells were spun at 150×g at 4°C for 5 min, and supernatant was decanted. This wash step was repeated two more times to total three washes. After decanting supernatant in the final wash, 1–3 drops of FACS buffer were added.
3
Collagen-Induced Arthritis in DBA/1J Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Arthritis was induced in DBA/1J mice between 8 and 10 weeks of age, as described previously33 (link). Chicken type II collagen (cCII; Sigma Chemical Co, St Louis, Missouri) was dissolved in 0.05 M acetic acid to a concentration of 4.0 mg/ml by overnight rotation at 4 °C and mixed with an equal volume of Freund's complete adjuvant (Wako Pure Chemical Industries, Ltd.). On day 0, DBA/1 J mice were immunized at the base of the tail with 100 μl of emulsion. The same injection was repeated on day 21. For CTLA-4 Ig treatment, mice were injected intravenously with 200 μg of CTLA-4 Ig (Bristol-Myers Squibb) labeled using a SAIVI Alexa Fluor 647 Antibody/Protein-Labeling Kit (S30044, Thermo Fisher Scientific), which can control the degree of labeling to prevent dispersion of the signal-to-background ratio, biodistribution, and clearance. We optimized the labeling conditions to achieve about four to five molecules of dye per antibody. Human IgG isotype control (02-7102, Thermo Fisher Scientific) was used for control experiments.
4
Surface Staining of Transfected HEK293FT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For surface staining, HEK293FT cells were plated at 3×10 5 cells per well in 1 mL DMEM in 12-well plates and transfected as described above, using 200 μL transfection reagent per well. At 36-48 h after transfection, cells were harvested with 500 µL FACS buffer and spun at 150×g at 4°C for 5 min. For the experiment in which multiple harvest methods were compared (Supplementary Fig. 14), some samples were harvested using 0.05% Trypsin-EDTA (3 min or 10 min incubation, 37°C), which were then quenched with medium and added to two volumes of FACS buffer. Supernatant was decanted, and 50 µL fresh FACS buffer and 10 µL human IgG (Human IgG Isotype Control, ThermoFisher Scientific #02-7102, RRID: AB_2532958, stock concentration 1 mg/mL) was added. Cells were incubated in this mixture at 4°C for 5 min. 5 µL FLAG tag antibody (Anti-DDDDK-PE, Abcam ab72469, RRID: AB_1268475, or Anti-DDDDK-APC, Abcam ab72569, RRID: AB_1310127) was added at a concentration of 0.5 µg per sample and cells incubated at 4°C for 30 min. Following incubation, 1 mL FACS buffer was added, cells were spun at 150×g at 4°C for 5 min, and supernatant was decanted. This wash step was repeated two more times to total three washes. After decanting supernatant in the final wash, 1-3 drops of FACS buffer were added.
5
Chromium-51 Release Assay for Cytotoxicity
6
Aducanumab Treatment in Aged Mice
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!