CTLA-4-EGFP-expressing cells were incubated in PBS containing 10% goat serum (Sigma–Aldrich) at room temperature for 15 min. CTLA-4-EGFP-expressing cells were then incubated with the purified anti-CTLA-4 mAbs (4G2-A3 and 4C2-D9) (0, 1.25, 2.5, 5, 10, and 20 μg/ml) or a mouse IgG1 isotype control (Southern Biotech) for 30 min at 37°C. The cells were then washed twice and incubated with 0.2 μg/ml CD80-Ig or 0.2 μg/ml CD86-Ig for 30 min at 37°C. CD80-Ig or CD86-Ig was labeled with Alexa Fluor 647-conjugated anti-rabbit IgG (H+L) goat IgG (Thermo Fisher Scientific) and analyzed using a FACSVerse. Three independent experiments were performed. The relative binding of CD80-Ig or CD86-Ig to CTLA-4-expressing cells was calculated using the value of the mean fluorescence intensity (MFI) of a sample preincubated with each antibody vs that of cells preincubated without antibody (MFI=1).
Mouse igg1 isotype control
Manufactured by Southern Biotech
Sourced in United States
The Mouse IgG1 isotype control is a laboratory reagent used as a negative control in experiments involving the detection or quantification of mouse IgG1 antibodies. It serves as a reference to distinguish specific antibody binding from non-specific background signals.
Automatically generated - may contain errors
Lab products found in correlation
Goat serum, by Merck Group (2 mentions)
Alexa fluor 647 conjugated anti rabbit igg h l goat igg, by Thermo Fisher Scientific (1 mentions)
Fc block, by BD (1 mentions)
Alexa fluor 647 conjugated anti mouse igg h l f ab 2, by Thermo Fisher Scientific (1 mentions)
Facs verse, by Thermo Fisher Scientific (1 mentions)
3 protocols using mouse igg1 isotype control
1
CTLA-4 Binding Kinetics Assay
2
ErbB3 Receptor Blocking Assay
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Cells were detached with 2 mM EDTA, centrifuged, and resuspended at a concentration of 5 × 106 cells per mL in 100 μL in a FACS buffer containing PBS, 2 mM EDTA, and 2.5% FBS. Cells were then placed on ice and treated for 5 min with 5 μg/mL Fc Block (cat# 553142, BD Biosciences, San Jose, CA, USA). Then, either the ErbB3 blocking antibody (cat# MS-303-PABX, Thermo Scientific, Fremont, CA, USA) or mouse IgG1 isotype control (cat# 0102-01 Southern Biotech, Birmingham, AL, USA) were added at a concentration of 10 μg/mL for 30 min, with mixing of the tubes by flicking every 10 min to ensure proper labeling. Samples were then centrifuged and washed three times in the FACS buffer to eliminate any unbound antibody. Cells were then labeled with a donkey anti-mouse Alexa-647 conjugated secondary antibody (cat# 715-605-151, Jackson Immunoresearch, West Grove, PA, USA) for 30 min. Samples were then washed and filtered in preparation for FACS analysis. A total of 1 × 104 cells per sample were analyzed using a DXP10 Calibur flow cytometer and sample data were processed using FlowJo.
3
Measuring Anti-CTLA-4 mAb Reactivity
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The reactivity of the purified anti-CTLA-4 mAbs was measured using flow cytometry of CTLA-4-EGFP-expressing cells, which were incubated in PBS containing 10% goat serum (Sigma–Aldrich) for 15 min at room temperature. The cells were incubated with anti-CTLA-4 mAbs (10 µg/ml) (4G2-A3 [37 (link)] and 4C2-D9) or a mouse IgG1 isotype control (Southern Biotech, Birmingham, AL, USA) for 30 min at 37°C. The cells were washed twice, incubated with Alexa Fluor 647-conjugated anti-mouse IgG (H+L) F (ab’)2 (Thermo Fisher Scientific) for 30 min at 37°C, washed twice, and analyzed using a FACSVerse.
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