BAL cells were blocked with 1% BSA–containing TruStain fcX (anti–mouse CD16/CD32) antibody (101319, BioLegend), followed by staining with antibodies. Antibodies used were rat anti–mouse CD45-PE (12-0451-82, eBioscience), LIVE/DEAD–eFluor 506 (65-0866, Invitrogen), rat anti–mouse CD11b–APC-Cy7 (101225, BioLegend), anti–mouse CD64–PE-Cy7 (139313, BioLegend), rat anti–mouse Ly6G–Alexa Fluor 700 (561236, BD Biosciences), rat anti–mouse Siglec F–APC (155507, BioLegend), rat anti–mouse Ly6C–eFluor 450 (48-5932-82, Invitrogen), and rat anti–mouse MHC II–PerCP-Cy5.5 (562363, BD Biosciences). A hierarchical gating strategy was used to represent the TRAMs as CD45+CD11b+/–Ly6G–CD64+Ly6C–Siglec Fhi and MDMs as CD45+CD11b+/–Ly6G–CD64+Ly6C–Siglec Flo. Data were acquired on a FACSAria II or LSR II (BD Biosciences) using BD FACS DIVA software (version 8.0.1). Data were analyzed using FlowJo (FlowJo LLC) software (version 10.5.0).
Trustain fcx anti mouse cd16 cd32
Manufactured by BioLegend
Sourced in United States
TruStain FcX anti-mouse CD16/CD32 is a laboratory reagent used to block Fc receptors on mouse cells. It binds to CD16 and CD32 to prevent non-specific antibody binding, improving the specificity of downstream cell staining or analysis.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (2 mentions)
Facsaria 2, by BD (1 mentions)
Rat anti mouse cd11b apc cy7, by BioLegend (1 mentions)
Live dead efluor 506, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse cd45 pe, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd64 pe cy7, by BioLegend (1 mentions)
Rat anti mouse siglec f apc, by BioLegend (1 mentions)
Rat anti mouse mhc 2 percp cy5, by BD (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Anti cd45 fitc, by Thermo Fisher Scientific (1 mentions)
Dispase, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Elastase, by Merck Group (1 mentions)
Anti cd31 fitc, by Thermo Fisher Scientific (1 mentions)
Sh800s cell sorter, by Sony (1 mentions)
Anti cd326 apc, by Thermo Fisher Scientific (1 mentions)
Facs canto 2 instrument, by BD (1 mentions)
3 protocols using trustain fcx anti mouse cd16 cd32
1
Identifying Tissue-Resident and Monocyte-Derived Macrophages
2
Lung Cell Isolation and Sorting
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Lung dissociation was performed as described previously with simple modification (18 (link)). The protease solution used for lung digestion in this study contained Collagenase Type I (450 U/mL; Sigma, C0130), Elastase (4 U/mL; Sigma, E1250), Dispase (5 U/mL; Sigma, D4693), and DNaseI (0.33 U/mL; Roche, 10104159001, Mannheim, Germany) dissolved in Dulbecco’s minimal essential medium/F12 (DMEM/F12) (Gibco, 11330021, Grand Island, NY, USA). The whole-lung suspension was blocked with TruStain FcX anti-mouse CD16/CD32 (BioLegend, 101319, San Diego, CA, USA) in the Dulbecco’s phosphate buffered saline (D-PBS) buffer with 10% fetal bovine serum (FBS), and then stained with anti-CD31-FITC (eBioscience, 11-0311-81), anti-CD45-FITC (eBioscience, 11-0451-81), and anti-CD326-APC (eBioscience, 17-5791-82). Fluorescence activated cell sorting (FACS) was performed on an SH800S Cell Sorter (Sony, San Jose, CA, USA).
3
Murine Spleen Cell Immunophenotyping
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The spleens were harvested from the mice (n = 4 per genotype). Cells were released by pressing the spleens under sterile metal screens and erythrocytes were lysed in cold ammonium chloride-based buffer. Fc receptors were blocked with TruStain FcX anti-mouse CD16/CD32 (cat# 101320; BioLegend, San Diego, CA), and surface antigens were stained with fluorescence-labeled antibodies to mouse CD3, CD4, CD8, B220/CD45, IgM, CD21/35, CD23, CD19, and CD138 (Additional file 2 ). Data acquisition and analysis were performed as before [28 (link)] using a FACSCanto II instrument and BD FACSDiva software (version 5.0).
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