The single‐cell suspension was blocked with mouse FcR blocking (Miltenyi Biotec) prior to surface staining. Fixable viability dye eFluor 520 (eBioscience) was used to exclude dead cells. For intracellular staining in T cells, after ex vivo stimulation with the leukocyte activation cocktail (BD Biosciences) for 5 hours, cells were fixed and permeabilized by using a fixation/permeabilization kit (BD Biosciences) followed by intracellular cellular staining after staining with surface antibodies. Data were acquired on BD FACS Calibur and analyzed by Flow‐Jo.
Mouse fcr blocking
Manufactured by Miltenyi Biotec
Mouse FcR blocking reagent is a laboratory product designed to block Fc receptors on mouse cells. This product helps prevent non-specific binding of antibodies to Fc receptors, which is important for various immunological assays and experiments involving mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (1 mentions)
Fixation permeabilization kit, by BD (1 mentions)
Leukocyte activation cocktail, by BD (1 mentions)
Efluor 520, by Thermo Fisher Scientific (1 mentions)
Pe anti mouse f4 80 antigen, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd11b apc, by Thermo Fisher Scientific (1 mentions)
Anti mouse ly6g fitc, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
4 protocols using mouse fcr blocking
1
Single-Cell Flow Cytometry with Intracellular Staining
2
GFP-HCT-116 Cell Line Characterization
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The GFP-HCT-116 cell line (Retroviral GFP transduction) was purchased from Nanjing Origin Biotechnology Co. Ltd. (Nanjing, Jiangsu, China). Antibodies for flow cytometry were as follows: Mouse FcR Blocking (Miltenyi, 130–092–575), CD45-PE-Vio770 mouse (Miltenyi, 130–105-462), Anti-Mouse F4/80 Antigen PE (eBioscience, 12–4801-80), Anti-Mouse CD11b APC (eBioscience, 17–0112-81), Anti-Mouse Ly-6G-FITC (eBioscience, 11–9668-82).
3
Humanized Antibodies Block BAG3 Binding
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Example 4
BAG3 binds to the surface of macrophages and we have showvn that binding is specifically inhibited by the murine anti-BAG AC-2 antibody that sequesters BAG3 protein. Here we tested the ability of chimeric and humanized variants of the antibody to block binding to the macrophage surface.
Materials and Methods
J774 A.1 cells (1×106/ml) were incubated with blocking solution (PBS containing 5% FBS/0.1% NaN3) and with FcR blocking mouse (Miltenyi Biotec-cod. 130-092-575) (1 μl/1×106 cells) for 30 minutes at 4° C. Then, 1×105 cells were incubated with FITC-rBAG3 protein (40 μg/ml) alone or in presence of anti-BAG3 mouse antibody (AC2) (3200 μg/ml) or murine IgG1 (3200 μg/ml), or with the chimeric and humanized variants of the antibody (3200 μg/ml), or human IgG (3200 μg/ml) in blocking solution, for 30 minutes on ice. After incubation, cells were washed with PBS and analyzed by flow cytometry.
Results
Flow cytometric evaluation of fluorescent BAG3 binding to the surface of J774 A.1 macrophage cell line. Data are reported as mean fluorescence intensity (
4
Multiparametric Flow Cytometry of T Cells
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Cell suspensions were Fc-blocked (FcR Blocking mouse, 130-092-575; Miltenyi), and dead cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit-AmCyan (L34957; Thermo Fisher Scientific, Waltham, MA). Then, cells were incubated for 20 minutes at 4°C in the dark with a cocktail of antibodies directed against CD3 (APC-Vio770, 130-119-793; Miltenyi), CD4 (FITC, 130-118-692; Miltenyi), CD8 (PE-Vio770, 130-119-123; Miltenyi), CD62L (APC, 130-112-837; Miltenyi), CD44 (PE, 130-118-694; Miltenyi), and PD1 (PE-CF594, 562523 BD). Samples were washed twice with phosphate buffered saline. Samples were acquired using LSRFortessa (BD) and analyzed with FlowJo v10.
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