A portion of the perfused right lung lobes was mechanically digested and incubated for 30 min at 37°C at 5% CO2 in RPMI 1640 medium (Gibco) supplemented with glutamine, Na-pyruvate, 2-ME, penicillin, streptomycin, 10% heat-inactivated FCS, collagenase D (Roche), and collagenase type VIII (Sigma-Aldrich). Single-cell suspensions of lungs were then prepared by meshing the lungs through 40-μm nylon cell strainers and red blood cell lysis. Viable cells were counted by trypan blue exclusion. Cells were blocked with anti-mouse CD16/CD32 mAb (BioLegend) and human Fc receptor blocking solution (BioLegend), and then surface stained with monoclonal antibodies purchased from BioLegend: anti-mouse CD45 (clone 30-F11) anti-human CD45 (clone HI30), anti-human CD3 (clone UCHT1), anti-human CD4 (clone OKT4), anti-human CD8 (clone SK-1), anti-human CD19 (clone HIB19), anti-human CD20 (clone 2H7), anti-human CD33 (clone WM53), anti-human CD66a/c/e/b (clone ASL-32 and G10F5), anti-human CD14 (clone M5E2), and anti-human CD11c (clone Bu15) for 30 min at 4°C. The samples were analyzed by flow cytometry using a BD FACS CANTO II in the biosafety level 3 and visualized using FlowJo software.
Anti mouse cd16 cd32 mab
Manufactured by BioLegend
The Anti-mouse CD16/CD32 mAb is a monoclonal antibody that binds to the mouse CD16 and CD32 receptors. CD16 and CD32 are FcγR receptors involved in the regulation of immune cell activation.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Collagenase type 8, by Merck Group (1 mentions)
Human fc receptor blocking solution, by BioLegend (1 mentions)
Anti human cd14 clone m5e2, by BioLegend (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Collagenase d, by Roche (1 mentions)
S3 sorter, by Bio-Rad (1 mentions)
Mojosort mouse pan b cell isolation kit 2, by BioLegend (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Blocking one histo, by Nacalai Tesque (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Bx53f, by Olympus (1 mentions)
4 protocols using anti mouse cd16 cd32 mab
1
Isolation and Characterization of Lung Immune Cells
2
Isolation and Sequencing of B Cells from Infected Mice
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Cells were isolated from mes-LN and BM on day 28 or later after infection and on day 10 after transfer and B cells were purified with the MojoSort™ Mouse Pan B Cell Isolation Kit II (BioLegend). For cells after transfer Fc receptors were blocked with anti-mouse CD16/CD32 mAb and the cells stained at 4°C for 30 min for CD45.2 (Biolegend). Cells were sorted for tdTomato+ and tdTomato- before transfer and CD45.2+ tdTomato+ or CD45.2+ Tomato- cells after transfer using the S3 sorter (Bio-Rad). The cells were sorted into 1 ml FCS, pelleted and immediately lysed with RLT-buffer (RNeasy-Kit; Qiagen). The lysate was stored at -80°C until RNA isolation. RNA was isolated using the RNeasy micro kit (Qiagen). The samples were prepared for sequencing and analyzed according to the procedures described in (31 (link)).Samples were sequenced in an illumina MiSeq machine using the MiSeq Reagent Kit v3 (600-cycle).
3
Splenic Myeloid Cell Isolation
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The spleen was removed and homogenized to isolate splenocytes. Splenocytes were incubated in red-blood-cell lysis buffer (ACK lysing buffer, Gibco) to remove erythrocytes. After incubation with anti-mouse CD16/CD32 mAb (BioLegend, 0.5 μg per million cells) to block the Fc receptor, cells were then stained with antigen-presenting cell-conjugated CD11b, fluorescein isothiocyanate-conjugated Ly6c and phycoerythrin-conjugated Ly6G (BioLegend) in autoMACS running buffer containing bovine serum albumin, EDTA, PBS and 0.09% azide (Miltenyl Biotec) for 30 min. After washing cells with autoMACS running buffer, stained cells were analysed by FACSAria2 (BD Bioscience) and Flowjo software (Tree Star). Ly6c expression was evaluated in CD11b+Ly6G− splenic monocytes (apparent b).
4
Immunohistochemical Analysis of Murine Tumours
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Frozen CMS5a and B16F10 tumour specimens embedded in O.C.T compound (Sakura Finetech) were sectioned at a thickness of 3 μm, air-dried for 2 h, fixed with ice-cold acetone for 15 min, and subjected to immunohistochemistry. After washing three times with PBS, the tissue slides were incubated at 4 °C in blocking solution (PBS supplemented with 1% BSA, 5% Blocking One Histo [Nacalai Tesque]) and 0.2 µg/mL anti-mouse CD16/CD32 mAb (Biolegend) for 30 min. The tumour sections on the slides were then dual-labelled with PE-conjugated and FITC-conjugated mAbs, or triple-stained with PE-conjugated CD140a, APC-conjugated Sca-1 and FITC-conjugated Thy-1.1-specific mAbs diluted with PBS supplemented with 1% BSA and 5% Blocking One Histo for 1 h at room temperature in a humidified chamber. After washing three times with PBS supplemented with 0.02% Tween-20, the slides were mounted in Prolong Gold antifade reagent with/without DAPI (Life Technologies), and observed by fluorescence microscopy (BX53F; Olympus Co. Ltd.; Tokyo, Japan) or confocal laser scanning microscopy (LSM780; Carl Zeiss, Oberkochen, Germany). The photographs from PE-, FITC- and DAPI- or PE-, APC- and FITC-stained tissue sections were merged by Photoshop elements software (Adobe Systems Software).
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