Cells in culture were examined for surface expression by flow cytometry with BD Biosciences LSRFortessa and the following antibodies: anti-HLA-A,B,C (Biolegend, Cat#311436), anti-CD73 (Biolegend, Cat#344004), anti-CD39(R&D systems, Cat#FAB4397A), anti-PD-L1(Biolegend, Cat#329718), anti-CD45 (Biolegend, Cat#368511) or isotype IgG control antibodies (Biolegend, Cat#400268, Biolegend, Cat#400114, R&D systems, Cat#IC002A, Biolegend, Cat#400326, Biolegend, Cat#400122). LMP2 Tetramer- SSCSSCPLSK was purchased from MBL. Cells resuspended in 100 μl PBS containing 3% FBS were stained by each antibody. FlowJo v10 was used to perform analysis of flow cytometry raw data.
Lsrfortessa
Manufactured by BioLegend
Sourced in United States
The LSRFortessa is a high-performance flow cytometer designed for advanced multi-parameter analysis. It features a compact, modular design and offers a wide range of excitation lasers and detection channels, enabling simultaneous measurement of multiple cellular parameters.
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Lab products found in correlation
4 protocols using lsrfortessa
1
Comprehensive Immunophenotyping of Cultured Cells
2
Isolation of Colonic Lamina Propria Cells
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Colonic lamina propria cells were isolated as we have previously described17 . Briefly, colons were dissected, and epithelial cells were stripped by agitation in 10 mM EDTA for 30 min at 37 °C. The remaining tissue was transferred to 50 ml conical tubes and digested using collagenase VIII (Sigma-Aldrich, C2139-5G) in HBSS (with Ca/Mg) for 30–45 min at 37 °C. Cells were then filtered through 70 µm cell strainers and washed twice in 1 × PBS. Cell viability and numbers were determined using an automated cell counter (TECAN), stained with antibody cocktails as indicated and analyzed using an LSRFortessa (BioLegend, San Diego, CA, USA). Data were analyzed by Flow Jo V.10 software.
3
Multiparametric Flow Cytometry of Murine Immune Responses
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Single suspension cells from spleens and lymph nodes collected 14 days after the second immunization were prepared and stimulated with 10 μg/ml peptides at 37°C for 5 h. Cells were stained with viability dye eflour780 in PBS for 15 minutes on ice followed by washes twice with PBS supplemented with 2% FBS. To detect cell surface antigens, cells were stained with fluorochrome-tagged antibodies, as shown in the following table for 15 minutes on ice. To detect intracellular cytokines or intranuclear transcription factors, cells were fixed and permeabilized using an intercellular cytokine staining kit (Biolegend) or a commercial transcription factor staining kit (eBioscience). All stained samples were run on LSRFortessa (Biolegend) and analyzed by FlowJo (TreeStar).
| Antibody | Company | Clone | Lot number |
|---|---|---|---|
| anti-Mouse CD4-APC | eBioscience | GK1.5 | 4329627 |
| anti-Mouse CD8a-PerCP/Cy5.5 | Biolegend | 53–6.7 | B219152 |
| anti-Mouse TNFα-PE | eBioscience | MP6-XT22 | 438513 |
| anti-Mouse Granzyme B-PE Cyanine7 | eBioscience | NGZB | 4281151 |
| anti-Mouse IFNγ-APC | eBioscience | XMG1.2 | 4289683 |
| anti-Mouse IFNγ-BV421 | Biolegend | XMG1.2 | B232596 |
| anti-Mouse CD3e-FITC | eBioscience | 145–2C11 | E00061-1632 |
| anti-Mouse IL-5-PE | eBioscience | TRFK5 | 12–7052-82 |
| anti-Mouse IL-13-eFlour710 | eBioscience | eBio13A | 46–7133-82 |
4
Cell Surface Marker Profiling
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Cells in culture were examined for surface expression by flow cytometry with BD Biosciences LSRFortessa and the following antibodies: anti-HLA-A,B,C (BioLegend, catalog no. 311436), anti-CD73 (BioLegend, catalog no. 344004), anti-CD39 (R&D Systems, catalog no. FAB4397A), anti-PD-L1 (BioLegend, catalog no. 329718), anti-CD45 (BioLegend, catalog no. 368511) or isotype IgG control antibodies (BioLegend, catalog no. 400268, BioLegend, catalog no. 400114, R&D Systems, catalog no. IC002A, BioLegend, catalog no. 400326, BioLegend, catalog no. 400122). LMP2 Tetramer-SSCSSCPLSK was purchased from MBL. Cells resuspended in 100 μL PBS containing 3% FBS were stained by each antibody. FlowJo v10 was used to perform analysis of flow cytometry raw data.
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