Lymphoid cells from bone marrow, thymus and spleen were isolated from 8-week old mice. To reveal T cell progenitor subsets in the thymus, single cell suspensions were stained with conjugated antibodies, specific for CD3-FITC (Biolegend), CD4-APC, CD8a-PerCp-Cy5.5, CD25-PE, CD44-APCCy7 (Biolegend), TCRβ-Pacific Blue (Biolegend). To analyse lymphoid subsets in the spleen, single cell suspensions were stained with CD3-FITC, CD4-APC, CD8a-PerCp-Cy5.5, CD19-APCH7, CD45R(B220)-PacificBlue, IgD-PE (eBioscience), IgM-PECy7 (eBioscience). To define the B cell progenitor subsets, suspensions from the bone marrow were stained with IgD-FITC, CD25-PE, IgM-PECy7 (eBioscience), CD45R (B220)-PacificBlue, CD117 (cKit)-APC (eBioscience), CD19-APCH7. Dead cells were excluded from the analysis by propidium iodide staining. Antibodies were purchased from BD Pharmingen unless mentioned otherwise. Samples were measured on a FACS Fortessa® and analysed using FlowJo® software (Version: 10.0.8r1).
Facs fortessa
Manufactured by FlowJo
The FACS Fortessa is a high-performance flow cytometer designed for advanced cell analysis. It features multiple laser configurations and a range of detection channels, enabling the simultaneous measurement of various cellular parameters.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 apc, by Thermo Fisher Scientific (1 mentions)
Cd3 fitc, by Thermo Fisher Scientific (1 mentions)
Cd25 pe, by Thermo Fisher Scientific (1 mentions)
Cd3 fitc, by BioLegend (1 mentions)
Cd4 apc, by BioLegend (1 mentions)
Cd25 pe, by BioLegend (1 mentions)
Cd44 apc cy7, by BioLegend (1 mentions)
Tcrβ pacific blue, by BioLegend (1 mentions)
Cd117 c kit apc, by Thermo Fisher Scientific (1 mentions)
Igd pe, by Thermo Fisher Scientific (1 mentions)
Igd fitc, by Thermo Fisher Scientific (1 mentions)
Cd8a percp cy5, by BioLegend (1 mentions)
Cd8a percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd19 apch7, by Thermo Fisher Scientific (1 mentions)
Cd45r b220 pacificblue, by Thermo Fisher Scientific (1 mentions)
Igm pecy7, by Thermo Fisher Scientific (1 mentions)
Click it edu alexa fluor 647 kit, by Thermo Fisher Scientific (1 mentions)
Sytox staining, by Thermo Fisher Scientific (1 mentions)
4 protocols using facs fortessa
1
Dissecting Murine Lymphoid Cell Subsets
2
Quantifying MCF-7 Cell Apoptosis
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Cell apoptosis was detected by a TransDetectTM Annexin V-EGFP/PI Cell Apoptosis Detection Kit (TRAN, China). Briefly, 2×105 MCF-7 breast cancer cells were added to the 6-well plates overnight, and MCF-7 cells were harvested after 6-TG treatment for 48 h. Subsequently, the cells were carefully washed with prechilled PBS 2 times and centrifuged at 500 g at 4°C for 5 min. Then, the cells were resuspended in 100 μL of prechilled 1× annexin V-binding buffer, and 5 μL of annexin V-EGFP and 5 μL of PI were added and reacted in the dark at room temperature for 15 min. Finally, 200 μL of 1× annexin V-binding buffer was added. The samples were detected by flow cytometry (BD, FACS Fortessa) and the cell apoptosis was analyzed using FlowJo software.
3
Cell Cycle Analysis of Irradiated Nestin+ Cells
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Click-iT EdU Alexa Fluor 647 kit was used (#C10424; Thermo Fisher Scientific). Briefly, MGG4 NestinP dTomato cells were seeded at an initial density of 1 × 105 cells/well in 6-well plates. Depending on time points and treatments employed (5 Gy irradiation or preconditioning with bEnd.3 CM), cells were harvested, resuspended as single cell using accutase, washed with PBS and incubated with EdU at a concentration of 10 μM for 2 h at 37 °C. EdU incorporation was subsequently detected by Alexa Fluor 647 azide, followed by Sytox staining as per manufacturer’s protocol (Thermo Fisher Scientific). Using FACS Fortessa and FlowJo 10, the fraction of cells in SubG1, G0/G1, S and G2/M phases were determined in each condition. The histogram plots for fraction of cells in each cell cycle phase was plotted using GraphPad 8 software.
FACS gating strategies provided in Supplementary Fig.17, 18 .
FACS gating strategies provided in Supplementary Fig.
4
Screening Compounds for CDX2 Induction
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A library of all compounds tested in the combinatorial screen can be found in Table S4. To identify positive modulators for CDX2 induction, CDX2-eGFP TSCs were cultured in TX medium for 24 h and then exposed to new TX media containing the different concentrations of individual compounds. After 48 h, CDX2 expressions of TSCs in each condition were analyzed by flow cytometry (FACS Fortessa, and FlowJo). For the experiment in Figure S3B, IGF2 (50 ng/mL) and ZSTK474 (PI3K inhibitor, 200 nM) were added to the medium.
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