Flow cytometry data were collected on a Gallios 10-color, 3-laser flow cytometer (Beckman Coulter) and analyzed with FlowJo software (Treestar). Cells were stained by standard protocols with the following antibodies (eBiosciences unless otherwise noted): Ly5.1 (A20, CD45.1), Ly5.2 (104, CD45.2), Ly6C/G (RB6-8C5, Gr-1), CD3e (145-2C11), CD45R (RA3-6B2, B220), CD11c (N418), TER-119, CD41 (MWReg30), CD117 (ACK2, c-kit), and Ly-6A/E (D7, Sca).
Ly5.1
Manufactured by Thermo Fisher Scientific
The Ly5.1 is a lab equipment product designed for specific applications. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (2 mentions)
Uea 1, by Vector Laboratories (1 mentions)
Anti foxp3 antibody, by Thermo Fisher Scientific (1 mentions)
Anti cd45 antibody, by Miltenyi Biotec (1 mentions)
Epcam, by BioLegend (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Liberase, by Roche (1 mentions)
Epcam g8, by BioLegend (1 mentions)
Mhcii, by BD (1 mentions)
Mhcii, by Thermo Fisher Scientific (1 mentions)
Epcam, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Liberase th, by Roche (1 mentions)
4 protocols using ly5.1
1
Multiparametric Flow Cytometry Analysis
2
Thymic Epithelial Cell Analysis Protocol
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For the analysis of thymic epithelial cells (TECs), minced thymuses were digested with 1 unit/ml Liberase (Roche) in the presence of 0.01% DNase I (Roche). Single-cell suspensions were stained with antibodies specific for CD326 (EpCAM; BioLegend), CD45 (eBioscience), and CD249 (Ly51, eBioscience), and for the reactivity with UEA-1 (Vector Laboratories). For the analysis of Aire and CCL21, surface-stained cells were fixed in 4% (g/vol) paraformaldehyde, permeabilized in 0.1% saponin, and stained with anti-Aire (5H12; eBioscience) antibody or anti-CCL21 (AAM27; Bio-Rad Laboratories) antibody. For the analysis of thymocytes, splenocytes, and lymph node cells, cells were surface-stained with the indicated antibodies. For the intracellular staining of Foxp3, the surface-stained cells were fixed and permeabilized by using a Foxp3 Staining Buffer Set (eBioscience) and stained with anti-Foxp3 antibody (eBioscience). For the isolation of TECs, CD45− cells were enriched in magnetic bead conjugated anti-CD45 antibody (Miltenyi Biotec). Multicolor flow cytometry and cell sorting were performed on FACSAria II (BD).
3
Identification and Isolation of Murine Medullary Thymic Epithelial Cells
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The thymi were harvested, washed with cold PBS twice, minced and digested with Liberase TH (Roche) and DNase I (Roche). Thymic cells were stained with antibodies: EpCAM (G8.8, Biolegend, 118204), Ly51 (Biolegend, 108312), MHCII (BD sciences, 562366), CD45 (eBioscience, 45-0451-82) for 30 min on ice. mTECs were gated as CD45−EpCAM+Ly51−MHCII+.
For Aire staining, cells were stained with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and stained with Anti-Aire antibody (eBioscience, 51-5934-82) for 30 min on ice.
For Aire staining, cells were stained with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and stained with Anti-Aire antibody (eBioscience, 51-5934-82) for 30 min on ice.
4
Multiparametric Flow Cytometry Analysis
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