For analysis of receptor expression, after removal from culture plates, cells were incubated with the primary antibody for 30 min at 0 °C. Primary antibodies: anti-mouse MHCII I-A/I-E (eBioscience); anti-mouse CD301 (AbD Serotec). Phagocytosis analysis was performed using 2 μm latex beads (Sigma). The beads were opsonized for 30 min at 37 °C with mouse serum IgG (Sigma) before adding to the cells and incubating for 30 min at 37 °C. Glucose uptake was assessed using 2NBDG fluorescent glucose (Invitrogen) [50 μM] for 30 min at 37 °C or 0 °C. Samples were analysed on a FACScalibur (BD). Granularity was detected in the SSC channel, and fluorescence was detected using an argon laser. Sorting of RAW264.7 cells for determination of cell doublets was performed using a FACSAria II.
Mouse serum igg
Manufactured by Merck Group
Mouse serum IgG is a laboratory reagent used in various immunological assays and applications. It is a purified immunoglobulin G (IgG) fraction isolated from the serum of mice. IgG is the most abundant type of antibody found in the blood and plays a crucial role in the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Lsr 2 flow cytometer, by BD (2 mentions)
Fab9332r, by R&D Systems (2 mentions)
Fab9333g, by R&D Systems (2 mentions)
Ic003r, by R&D Systems (2 mentions)
Ic003g, by R&D Systems (2 mentions)
Facscalibur, by BD (1 mentions)
2nbdg fluorescent glucose, by Thermo Fisher Scientific (1 mentions)
Recombinant ifn γ, by Thermo Fisher Scientific (1 mentions)
Clodronate liposome, by Yeasen (1 mentions)
Ivermectin, by MedChemExpress (1 mentions)
Af 488 mouse anti human ace2, by R&D Systems (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Papain, by Merck Group (1 mentions)
Affi gel protein a maps 2 kit, by Bio-Rad (1 mentions)
Cysteine, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid disodium salt, by Merck Group (1 mentions)
5 protocols using mouse serum igg
1
Immunophenotyping and Functional Assays for Macrophages
2
Preparation and Characterization of Aggregated IgG
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Antibodies used were listed in Suppl. Table 2 . Fludarabine monophosphate (TCI Chemicals) and ivermectin (MedChemExpress) were freshly prepared by dissolving in DMSO at high concentrations and further diluted in saline (5% DMSO) for injection. Clodronate liposomes and PBS liposomes were obtained from Yeasen. Recombinant IFN-γ was purchased from PeproTech. Aggregated IgG (AIgG) was prepared by heating mouse serum IgG (Sigma) at 62 °C for 30 min and centrifuging at 18000 × g for 20 min.
3
Quantifying Human ACE2 Expression
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Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (Clone # 535919) (R&D systems, FAB9332R), AF-488 mouse anti-human ACE2 (Clone # 171607) (R&D systems, FAB9333G) (0.4 µg/106 cells), AF-647 isotype control mouse IgG2b (Clone # 20102) (R&D systems, IC003R) or AF-488 isotype control mouse IgG2bAF488 (Clone # 20102) (R&D systems, IC003G) for 45 min on ice, followed by washing twice with 2% EV-free FBS/PBS. Finally, the cells were diluted in 2% EV-free FBS/PBS and analyzed on a BD-LSR II flow cytometer (BD Biosciences). Data were analyzed by BD FACSDiva softwares v8.0.2 or v8.0.3 or Flow Jo v10.6.2.
4
Purification and Fragmentation of IgG
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R6.5 IgG in culture media were affinity purified from cell culture supernatants using Affi-Gel Protein A MAPS II Kit (Bio-Rad). Eluted fractions were immediately neutralized, concentrated using 10 kDa cut-off Amicon Ultra-15 Centrifugal Filter Units (EMD Millipore), and buffer exchanged into PBS using PD-10 Desalting Columns (GE Healthcare). Fab fragments of R6.5 and mouse serum IgG (Sigma-Aldrich) were generated by digesting IgG with papain [35 ]. One volume of 2 mg/ml IgG in PBS, pH 6.2, was incubated at 37°C for 15 h with one volume of 20 μg/ml papain (Sigma-Aldrich) in PBS containing 20 mM ethylenediaminetetraacetic acid disodium salt (Sigma-Aldrich) and 20 mM cysteine (Sigma-Aldrich), pH 6.2. Fab fragments were separated from Fc and undigested IgG by Affi-Gel Protein A MAPS II Kit, followed by buffer exchange into PBS using PD-10. The concentration of antibody and fragments was determined by measurement with a NanoDrop (Thermo Scientific). Antibody and digestion products were analyzed on SDS-PAGE.
5
ACE2 Expression Analysis by Flow Cytometry
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Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (R&D systems, FAB9332R), AF-488 mouse anti-human ACE2 (R&D systems, FAB9333G), AF-647 isotype control mouse IgG2b (R&D systems, IC003R) or AF-488 isotype control mouse IgG2bAF488 (R&D systems, IC003G) for 45 min on ice, followed by washing twice with 2% exosome-free FBS/PBS. Finally, the cells were diluted in 2% exo-free FBS/PBS and analyzed on a BD-LSR II flow cytometer (BD Biosciences).
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