Splenocytes were harvested from groups of mice and transferred to a 96-well V-bottom plate. The following anti-mouse surface antibody markers (shown with clones, BioLegend) were used to stain the cells: CD19 (6D5, Alexa Fluor® 488), B220 (RA3-6B2, Alexa Fluor® 700), IgM (RMM-1, APC/Cyanine7), IgD (11-26c.2a, Pacific Blue™), CD95 (SA367H8, PE), GL7 (GL7, PE/Cy7) and CD4 (GK1.5, Brilliant Violet 421™). Cells were incubated in the dark on ice for 15 min and washed twice. For transcription factor staining, True-Nuclear™ Transcription Factor Staining Protocol was followed (BioLegend). Briefly, True-Nuclear™ 1× Fix Concentrate was added to the cells and incubated for 1 h in the dark at RT, after which cells were washed using True-Nuclear™ 1× Perm Buffer. This was followed by the addition of anti-mouse transcription factor-specific antibody, anti-Forkhead box P3 (FoxP3) (150D, Alexa Fluor® 647), and incubation in the dark at RT for 30 min. Finally, cells were washed with perm buffer, and resuspended cells were acquired using the CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA, USA).
True nuclear transcription factor staining protocol
Manufactured by BioLegend
The True-Nuclear™ Transcription Factor Staining Protocol is a laboratory technique used to detect and quantify the expression of transcription factors within the nucleus of cells. It provides a reliable method for the analysis of these critical regulatory proteins.
Automatically generated - may contain errors
Lab products found in correlation
Cytoflex lx flow cytometer, by Beckman Coulter (1 mentions)
Cell staining buffer, by BioLegend (1 mentions)
Alexa fluor 647anti mouse foxp3 catalog 126408, by BioLegend (1 mentions)
Ammonium chloride solution, by STEMCELL (1 mentions)
Cell surface immunofluorescence staining protocol, by BioLegend (1 mentions)
Red blood cell lysis buffer, by Abcam (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
3 protocols using true nuclear transcription factor staining protocol
1
Comprehensive Murine Lymphocyte Phenotyping
2
Isolation and Identification of Murine CD4+ Treg Cells
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Splenocytes were freshly isolated from the spleens of recipient mice. Briefly, the spleen was mashed through a cell strainer and centrifuged at 1000 rpm for 5 min. Then, the cells were washed in cell staining buffer (BioLegend) and centrifuged at 1400 rpm for 5 min. The red blood cells were lysed by ammonium chloride solution (Stemcell) for 10 min, followed by washes and centrifugation. Finally, the cells were stained by FITC anti-mouse CD4 (Catalog #100509) according to the Cell Surface Immunofluorescence Staining Protocol (BioLegend), followed by staining with Alexa Fluor 647 anti-mouse FOXP3 (Catalog #126408) according to True-Nuclear™ Transcription Factor Staining Protocol (BioLegend). After that, the percentage of CD4+ Foxp3+Treg cells was evaluated by flow cytometry.
3
Comprehensive Immune Cell Profiling
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The immune cell populations (CD3 + T cells, CD4 + T cells, CD8 + T cells, regulatory T cells [CD4 + CD25 + FoxP3 + Tregs], myeloid-derived suppressive cells [CD11b + Gr-1 + MDSCs]) in spleens and tumor tissues with different treatments were analyzed by flow cytometry. Cell suspensions from spleens or tumor tissues were filtered with a nylon net (90 × 90 mm; Dakewe Biotech, Shenzhen, China) and red blood cells were lysed with Red Blood Cell Lysis Buffer (Catalog #5831-100; Biovision, CA, USA). The immune cells from tumor tissues were separated with a kit (WBC1092Z; HaoYang Biological, Tianjin, China).
For membrane staining, cells were incubated with combinations of antibodies (CD3, CD4, CD8, CD11b, Gr-1 and CD25). For intracellular staining for the antibody FoxP3, cells were fixed and permeabilized immediately after cell surface staining in accordance with the True-Nuclear™ Transcription Factor Staining Protocol (Biolegend). All antibodies were purchased from Biolegend and flow data were collected on a Gallios flow cytometer (Beckman Coulter, CA, USA). The data were analyzed using FlowJo 6 software (Tree Star).
For membrane staining, cells were incubated with combinations of antibodies (CD3, CD4, CD8, CD11b, Gr-1 and CD25). For intracellular staining for the antibody FoxP3, cells were fixed and permeabilized immediately after cell surface staining in accordance with the True-Nuclear™ Transcription Factor Staining Protocol (Biolegend). All antibodies were purchased from Biolegend and flow data were collected on a Gallios flow cytometer (Beckman Coulter, CA, USA). The data were analyzed using FlowJo 6 software (Tree Star).
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