Following stimulation, cells were washed with PBS and stained with the Fixable Viability Dye Zombie Near-IR (BioLegend) for 15 minutes at room temperature. Samples were then surface stained for 30 minutes at room temperature. For the ICS assay this included: anti-CD3 BV605, anti-CD4 BV570, and anti-CD8 PerCP-Cy5.5. For the proliferation assay: anti-CD3 BV605, anti-CD4 BV786, anti-CD8 PerCP-Cy5.5, and anti-TCR γδ BV480. Following the surface stain, cells were fixed and permeabilized on ice for 1 hour using the FoxP3 Transcription Staining Buffer Set (eBioscience). Cells were then stained for intracellular markers on ice for 40 min. For the ICS assay this included: anti-IFNγ Alexa Fluor 700, anti-TNFα Alexa Flour 647, anti-IL-4 PE-Dazzle594, and anti-IL-13 FITC. For the proliferation assay this included: anti-IFNγ Alexa Fluor 700, anti-TNFα Alexa Flour 647, and anti-IL-4 PE-Dazzle594. Finally, cells were washed in permeabilization buffer and resuspended in PBS. Samples were acquired using a BD LSR II flow cytometer. 6 peak Rainbow Calibration Particles (BioLegend) were used to standardize instrument settings.
Foxp3 transcription staining buffer set
Manufactured by Thermo Fisher Scientific
The FoxP3 Transcription Staining Buffer Set is a laboratory tool designed for the detection and analysis of FoxP3-expressing cells. It provides the necessary reagents to perform intracellular staining and flow cytometric analysis of FoxP3, a transcription factor that is critical for the development and function of regulatory T cells.
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Lab products found in correlation
Fixable viability dye zombie near ir, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
6 peak rainbow calibration particles, by BioLegend (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Anti acetyl histone h3 lys27, by Cell Signaling Technology (1 mentions)
Anti acetyl histone h3 lys9, by Cell Signaling Technology (1 mentions)
Anti histone h3 d1h2, by Cell Signaling Technology (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Lsrfortesa, by BD (1 mentions)
Facsaria instrument, by BD (1 mentions)
Lsrii apparatus, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
7 protocols using foxp3 transcription staining buffer set
1
Flow Cytometric Analysis of T Cell Responses
2
Tracking Salmonella-specific CD4 T Cells
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Tracking Salmonella specific CD4 T cell response using MHC class II tetramers was performed as described previously [17 (link)]. Malaria-infected or control mice were injected i.v. with lysates of 108 CFU heat-killed Salmonella. Four hours later, spleens and livers were collected and processed into single cell suspensions. Cells were stained with PE-conjugated 2W1S::I-Ab tetramer in the presence of Fc block (culture supernatant from the 2.4G2 hybridoma, 2% mouse serum, 2% rat serum) room temperature for one hour. After washing with 2% FBS/PBS, cells were strained with fluorochrome-conjugated antibodies specific for CD3, CD4, CD8, and CD44 (eBioscience, San Diego, CA). For intracellular cytokine staining, cells were surface stained, and then treated with Foxp3/Transcription Staining buffer set (eBioscience) according to the manufacturer’s recommendation. Permeabilized cells were stained with fluorochrome-conjugated antibodies specific for IFN-γ (eBioscience). Cells were then analyzed by flow cytometry using a BD LSR Fortessa (BD Biosciences). All data sets were analyzed using FlowJo software (Tree star, San Carlos, CA).
3
Intracellular Staining and Flow Cytometry
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Single cell suspensions were stained for surface markers in PBS for 20 minutes at 4°C. Intracellular proteins (i.e., cytokines, FOXP3, and Ki67) were assessed using the FOXP3/Transcription staining buffer set (eBioscience, San Diego, CA) and following manufacturer’s instructions. Cells were permeabilized for 30 minutes and stained for intracellular proteins for 1 hour at 4°C. All fluorochromes were purchased from Biolegened and eBioscienes. Ex vivo re-stimulation was performed using PMA (Sigma-Aldrich), ionomycin (Sigma-Aldrich), and Golgi Stop (BD Biosciences) for 4 hours as previously described (Kornete et al., 2012 (link)). For OTI and OTII peptide re-stimulation, peptides OVA257-264 and OVA323-339, were incubated with transgenic T cells at 10μg/ml with Golgi Stop (BD Biosciences) for 6 hours followed by surface staining and ICS. Dead cells were distinguished using the fixable viability dye efluor780® from eBioscience. Single cell suspensions were fixed and permeabilized using the FOXP3 Transcription staining buffer set. Samples were acquired on the BD LSRFortessa™ and on the BD FACSymphony™. Flow cytometry data was analyzed using FlowJo (TreeStar).
4
Characterizing Immune Cell Epigenetics
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Mice trained in vivo by injection of β‐glucan (25 mg kg−1) and untrained mice, as above, were sacrificed after 7 d. Their lungs were isolated and treated with collagenase and dispase to yield single‐cell suspensions. After lysing RBCs, the cells were fixed with by incubation with CellCover solution (Anacyte Laboratories, 800‐125) for 10 min at room temperature and centrifuged at 500 x g for 5 min. After discarding the supernatant, cell fixation and permeabilization solution (Invitrogen, 005523‐00) was added to each pellet, followed by vortexing and incubation for 1 h at room temperature or 4 °C. The cells were washed, and staining buffer (eBioscience Foxp3/Transcription staining buffer set) was added, followed by the addition of antibodies (1:50) and incubation for 30 min at room temperature. These antibodies included anti‐histone H3 (D1H2, 12167S), anti‐tri‐methyl‐histone H3 (K4) (C42E8, 12064S), anti‐acetyl‐histone H3 (Lys 9) (C5B11, 4484S), and anti‐acetyl‐histone H3 (Lys27) (D5E4, 15485S), all from Cell Signaling. Cells were classified as epithelial cells by their expression of EpCAM and as macrophages by their expression of Siglec F. After washing, these cells were subjected to flow cytometry.
5
Multiparametric Flow Cytometry Analysis
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Cells were collected, washed in PBS, and stained with a viable dye (Zombie Aqua, Biolegend, #423101) for 20 min on ice in the dark. Cells were then resuspended in Fc‐blocking solution (anti CD16/CD32 antibody 2.4G2, homemade), incubated for 5 min and then labeled with indicated antibodies for 15 min at 4°C in the dark. Cells were then washed and fixed for 30 min to ON at 4°C (Foxp3/Transcription Staining Buffer Set, eBioscience, #00‐5523‐00). Fixed cells were permeabilized using the same buffer set and intracellular stainings were performed for 30 min at 4°C in the dark. Stained cells were collected and measured on a BD FACSCanto3 machine. Analyses were performed using the FlowJo software. The list of antibodies adopted in the study is provided in Appendix Table S1 .
6
Lymphocyte Profiling by Flow Cytometry
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Phenotyping of lymphocyte populations was performed by flow cytometry after preparation of single cell suspensions from lymphoid organs and staining using antibodies listed in Table S6. Single cell suspension of thymus, spleen and lymph nodes were prepared in FACS buffer (2% FBS, 1 mM EDTA, 1% penicillin-streptomycin, in PBS) by tissue homogenization with a syringe plunger against a 40 mm cell strainer. For preparation of cell suspensions from bone marrow, femur, tibia and pelvis were flushed with FACS buffer using a 10 mL syringe and a 26 gauge-needle and then passed through 40 mm cell strainer to obtain single cell suspensions. To achieve red blood cell lysis, the cell suspensions were treated with ACK lysis buffer (0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA in H2O, pH 7.2-7.4), washed and resuspended in FACS buffer. For intracellular staining the cells were first stained with a fixable viability dye prior to surface antibody staining. After cell surface staining the cells were fixed and permeabilized using the FoxP3/Transcription staining buffer set (eBioscience) according to manufacturer's protocol followed by intracellular antibody staining. Data were collected on an LSRFortesa and/or LSRII apparatus (BD Biosciences) and were analyzed with FlowJo software version 10; cell sorting was done using a FACSAria instrument (BD Biosciences).
7
Multiparameter Flow Cytometry Analysis
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Following stimulation, cells were washed with PBS and stained with the Fixable Viability Dye Zombie Near-IR (BioLegend) for 15 min at room temperature. Samples were then surface stained for 30 min at room temperature. For the ICS assay this included: anti-CD3 BV605, anti-CD4 BV570, anti-CD8 PerCP-Cy5.5, anti-CCR4 BV421, and anti-CXCR3 BV711. For the proliferation assay: anti-CD3 BV605, CD4 BV786, anti-CD8 PerCP-Cy5.5, anti-CCR4 BV421, and anti-CXCR3 BV711. Following the surface stain, cells were fixed and permeabilized on ice for 1 h using the FoxP3 Transcription Staining Buffer Set (eBioscience). Cells were then stained for intracellular markers on ice for 40 min. For the ICS assay this included: anti-T-bet PE-Cy7, anti-GATA3 PE, anti-IFN-γ Alexa Fluor 700, anti-TNFα Alexa Flour 647, anti-IL-4 PE-Dazzle594, and anti-IL-13 FITC. For the proliferation assay this included: anti-T-bet PE-Cy7, anti-GATA3 PE, anti-IFN-γ Alexa Fluor 700, anti-TNFα Alexa Flour 647, and anti-IL-4 PE-Dazzle594. Finally, cells were washed in permeabilization buffer and resuspended in PBS. Samples were acquired using a BD LSR II flow cytometer.
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