An adult RosaYFP/YFP mouse received retro-orbital injection of AAV-TBG-cre at the dose of 2.5 × 1011 viral particles, and hepatocytes were harvested 3 weeks later using a standard two-step collagenase perfusion method. Isolated RosaYFP/YFP hepatocytes were stained with APC anti-mouse CD326/EPCAM) (1:100, G8.8, Biolegend) or APC anti-human/mouse CD49f/Itga6 antibody (1/100, GoH3, Biolegend). For staining with Prom1/Cd133 and Cd24, cells were incubated with purified rat anti-mouse CD133/Prom1 antibody (1:100, 315–2 C11, Biolgend) and rat anti-mouse CD24 antibody (1:100, Biolegend, M1/69) followed by staining with Alexa647-conjugated donkey anti-rat antibody (1:300, Jackson ImmunoResearch). APC-conjugated rat IgG2a or IgG2b, κ isotype control antibody was used as control. DAPI was added to stain dead cells. Attune NxT Flow Cytometer (Lifetechnologies) was used for data collection. B6 wild-type adult hepatocytes, which were stained with only DAPI, was used for making the threshold of YFP signal.
M1 69
Manufactured by BioLegend
The M1/69 is a mouse monoclonal antibody that recognizes the CD34 antigen. CD34 is a type I transmembrane sialoglycoprotein expressed on hematopoietic stem and progenitor cells, as well as on vascular endothelial cells. The M1/69 antibody can be used for the identification and isolation of CD34-positive cells.
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Lab products found in correlation
M5 114, by BioLegend (4 mentions)
Facsaria 2, by BD (2 mentions)
Fjk 16, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
H57 597, by BioLegend (2 mentions)
Michel 19, by BioLegend (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Fixation permeabilization kit, by Thermo Fisher Scientific (2 mentions)
Cd11c hl3, by BD (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Apc anti mouse cd326 epcam, by BioLegend (1 mentions)
Cd24 fitc, by BioLegend (1 mentions)
Facscelesta flow cytometer, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Cd49f pe, by BD (1 mentions)
Fortessa x20, by BD (1 mentions)
Live dead near ir stain, by Thermo Fisher Scientific (1 mentions)
X54 5 7, by BioLegend (1 mentions)
M1 70, by BioLegend (1 mentions)
Ra3 6b2, by BioLegend (1 mentions)
7 protocols using m1 69
1
Isolation of Rosa YFP Hepatocytes
2
Epithelial Cell Phenotyping by Flow Cytometry
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Before and after purification, cells were resuspended in PBS with 5% FBS and stained using CD24-FITC (1:300, M1/69, BioLegend) and CD49f-PE (1:30, GoH3, BD Biosciences). Antibodies were incubated for 30 min at 4°C and then cells were washed twice in PBS + 5% FBS. Data were acquired using a BD FACSCelesta™ Flow Cytometer and BD FACSDiva™ software version 8.0.1.1 (Becton, Dickinson and Company). Data analyses were performed using FlowJo™ version 10.7.1 (Becton, Dickinson and Company, 2019). Ten thousand events were acquired for each sample. Cells were first gated by their forward and side scatter, representing cell size and granularity. Luminal and basal epithelial cells were distinguished respectively using CD24 and CD49f markers.
3
Flow Cytometric Characterization of Murine Lung Cells
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Single-cell suspensions from lungs were prepared as described in the previous section. Cells were stained for a panel of murine cell surface markers and analyzed using a Fortessa X-20 (BD) and FlowJo software (Tree Star). Antibody clones used included RM4-5 (anti-CD4), 53–6.7 (anti-CD8α), and H57-597 (anti-TCRβ chain), all obtained from BD. In addition, H1.2F3 (anti-CD69), IM7 (anti-CD44), XMG-6 (anti-IFN-γ), N418 (anti-CD11c), M5/114.15.2 (anti-IA/IE), HK1.4 (anti-Ly6C), 1A8 (anti-Ly6G), M1/70 (anti-CD11b), 2E7 (anti-CD103), P84 (anti-CD172α), X54-5/7.1 (anti-CD64), BM8 (anti-F4/80), M1/69 (anti-CD24), RA3-6B2 (anti-B220), 4B12 (anti-CCR7), MP1-22E9 (anti-CSFR2α), AFS98 (anti-CD115), 323 (anti-CD40), and GL-1 (anti-CD86) were obtained from BioLegend. Clone 475301 (anti-CCR2) was obtained from R&D Systems. Live/Dead Near IR stain was obtained from Life Technologies and included in all staining protocols. Optimal antibody concentrations were determined in separate experiments, and appropriate fluorochrome-labeled isotype control antibodies used throughout. In all cases, cells were first gated on singlets (FSC-W vs. FSC-A or -H) and live cells (Live/Dead Near IR negative) before further analyses.
4
Comprehensive Thymocyte and TEC Profiling
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Thymocytes were incubated with Abs against TCR beta (1:400, H57-597; Biolegend), CD4 (1:1000, GK1.5; BioLegend), CD5 (1:400, 53-7.3, eBioscience), CD8 (1:500, 53-67; BioLegend), CD25 (1:1000, PC61.5; BioLegend), CD44 (1:500, IM7; BioLegend), c-kit (1:200, 2B8; BioLegend), CD24 (1:1000, M1/69; BioLegend), CD69 (1:200, H1.2F3; BioLegend), PD-1 (1:200, 29F.1A12, Biolegend), CCR7 (1:200, 4B12, BioLegend). For intracellular staining, cells were fixed, permeabilized (Fixation/Permeabilization kit, eBioscience) and stained for Foxp3 (1:70, FJK-16S, eBioscience) and Helios (1:70, 22F6, eBioscience). TECs were stained using Abs against CD45 (1:400, 30F11; BioLegend), EpCAM (1:1000, G8.8; BioLegend), MHCII (1:1000, M5/114.15.2; BioLegend), Ly51 (1:1000, 6C3; BioLegend), UEA-1 (1:1000, Reactolab), CD80 (1:500, 16-10A1, Biolegend), CD86 (1:500, GL-1, Biolegend), CD83 (1:500, Michel 19, Biolegend). For intracellular staining, cells were fixed, permeabilized (Cytofix/Cytoperm Kit, BD Biosciences) and labelled for the expression of CD83. Stained samples were acquired on a FACSAria II or BD LSRFortessa flow cytometers and the data was analyzed using the FlowJo (Treestar) software.
5
Comprehensive Thymocyte and TEC Profiling
6
Isolation and Analysis of Intestinal Immune Cells
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MLN were processed into single cell suspensions and colons were digested and LP lymphocytes were isolated as described.24 For experiments requiring detection of intracellular antigens, cell suspensions were cultured in the presence of Golgistop (5 μL/mL; BD), phorbol myristate acetate (50 ng/mL; Sigma) and ionomycin (1mg/mL Sigma). Cells were then permeabilized and fixed using an intracellular staining kit (eBioscience). The following antibodies were used: CD4 (RM4-5, eBioscience), CD8α (53–6.72, Biolegend), CD11b (M1/70, Biolegend), CD45R/B220 (RA3-6B2, BD Pharmingen), CX3CR1 (SA011F11, Biolegend), CD11c (HL3, BD), MHCII (M5/114, Biolegend), CD24 (M1/69, Biolegend), CD64 (X54-5/7.1, Biolegend), CD103a (2E7, Biolegend), IL-17A (TC11-18H10, Biolegend), Foxp3 (FJK-16s, eBioscience) and IFNγ (XMG1.2, BD Pharmingen).
7
Multicolor Flow Cytometry for Dendritic Cell Subsets
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For FACS analysis as well as FACS sorting of single-cell suspensions, cells were first incubated with CD16/CD32-specific antibody (2.4G2; BD) for 10 min to block non-specific Fc-receptor interactions. A live death staining was performed using zombie aqua (eBioscience) according to the manufacturer’s instructions. To differentiate between DC subsets, cells were stained for 15 min at 4°C with combinations of antibodies specifically binding to CD11b (M1/70.15; Invitrogen), CD11c (HL3; BD), Siglec-H (eBio440c; eBioscience), CD69 (H1.2F3; BD), Clec9a (42D2; eBioscience), CD172 (SIRPα) (P84; Biolegend) and CD24 (M1/69; Biolegend). The cells were subsequently washed with 1 mL FACS buffer (PBS, 1% FCS) and then re-suspended in FACS buffer supplemented with 3% paraformaldehyde (PFA). Samples were measured using a FACS LSR II, and data were analyzed with FlowJo 7.6.5 software (TreeStar). Dead cells as well as cell duplets were excluded prior to subsequent analysis. DC subsets were sorted using the FACS Aria or FACS XDP and sorting efficiency ranged between 71.9–86.2% for the pDC, 81.4–94.4% for the CD11b-like DC and 44.54–83.2% for the CD8α-like DC.
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