Freshly isolated splenocytes were stained with PE-Cy5 anti-CD4 and PE-anti-CD25 or isotypes rat IgG2a,κ and rat IgM,κ, respectively, for 10 min at 4 °C (BD). Cells were then fixed and permeabilized using the Mouse FoxP3 Buffer Set and then incubated with Alexa Fluor 488 anti-FoxP3 or isotype rat IgG2baccording to manufacturer’s instructions (BD). Flow cytometry was performed immediately or within 24 h of permeabilization using a FACScaliber machine. 1 × 105 gated events were recorded. Results were analyzed with FCS Express software.
Anti foxp3 alexa fluor 488
Manufactured by BD
Sourced in United States
Anti-Foxp3-Alexa Fluor 488 is a fluorescently-labeled antibody that binds to the Foxp3 transcription factor, which is a marker for regulatory T cells. The Alexa Fluor 488 dye provides green fluorescence for detection and analysis using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Isotype rat igg2baccording, by BD (2 mentions)
Pe cy5 anti cd4, by BD (2 mentions)
Anti cd25 pe, by BD (2 mentions)
Mouse foxp3 buffer set, by BD (2 mentions)
Anti cxcr5 apc, by BioLegend (2 mentions)
Anti cd3 v450, by BD (2 mentions)
Transcription factor buffer, by BD (2 mentions)
Anti cd45ra pe cy7, by BD (2 mentions)
Facslyric system, by BD (2 mentions)
Anti pd 1 pe cy7, by BD (2 mentions)
Anti cd4 v500, by BD (2 mentions)
Perm wash buffer, by BD (2 mentions)
Anti cd4 apc, by BD (1 mentions)
Anti il 10 apc, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Cytofix cytoperm tm fixation permeabilization solution kit, by BD (1 mentions)
Anti pd 1 bv421, by BD (1 mentions)
Anti cd25 apc cy7, by BD (1 mentions)
Anti cd69 pe cy7, by BD (1 mentions)
Anti cd3 percp cy5, by BD (1 mentions)
6 protocols using anti foxp3 alexa fluor 488
1
Regulatory T Cell Phenotyping by Flow Cytometry
2
Murine Regulatory T Cell Phenotyping
3
Phenotypic Analysis of Immune Cells
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Fresh PBMCs from patients and healthy donors were washed and stained with the following antibodies at 4 °C for 15 min: anti-CD3-V450 (#560366, BD Biosciences), anti-CD4-V500 (#560769, BD Biosciences), anti-CD45RA-PE-Cy7 (#560675, BD Biosciences), anti-CXCR5-APC (#356907, BioLegend), and anti-PD-1-PE-Cy7 (#561272, BD Biosciences). For intracellular staining of anti-Foxp3-Alexa Fluor 488 (#560047, BD Biosciences), the cells were fixed and permeabilized with Transcription Factor Buffer at 4 °C for 30 min and were washed with Perm/Wash Buffer (BD Biosciences) before intracellular staining. Isotype-matched control antibodies were used to monitor the background. The well-stained cells were subjected to flow cytometry using a BD FACSLyric system and were further analyzed using the FlowJo v10 software (TOMY Digital Biology).
4
Multiparametric Flow Cytometry Analysis
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For surface staining, cells were stained with anti-CD3 PerCP-Cy5.5, anti-CD4 APC, anti-CD25 APC-Cy7, anti-CD69 Pe-Cy7, anti-PD-1 BV421, and anti-CD38 Alexa Fluor 488 (all from BD Biosciences, San Diego, CA, USA). Cells were then incubated for 15 minutes under room temperature and were fixed with 1% formaldehyde for 15 minutes. For intracellular staining, cells were permeabilized (Cytofix/Cytoperm TM Fixation/Permeabilization Solution Kit, BD Biosciences) for 40 minutes and stained with anti-FoxP3 Alexa Fluor 488, anti-CTLA-4 BV605, and anti-IL-10 APC. All the samples were analyzed using an LSRFortessa flow cytometer (Becton-Dickinson, San Jose, CA, USA) and analyzed by FlowJo software (FlowJo, Tree Star, Ashland, OR, USA).
5
Multicolor Flow Cytometry of PBMCs
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Fresh PBMCs from patients and healthy donors were washed and stained with the following antibodies at 4 ºC for 15 min: anti-CD3-V450 (#560366, BD Biosciences), anti-CD4-V500 (#560769, BD Biosciences), anti-CD45RA-PE-Cy7 (#560675, BD Biosciences), anti-CXCR5-APC (#356907, BioLegend), and anti-PD-1-PE-Cy7 (#561272, BD Biosciences). For intracellular staining of anti-Foxp3-Alexa Fluor 488 (#560047, BD Biosciences), the cells were xed and permeabilized with Transcription Factor Buffer at 4 °C for 30 min, and were washed with Perm/Wash Buffer (BD Biosciences) before intracellular staining. Isotype-matched control antibodies were used to monitor the background. The well-stained cells were subjected to ow cytometry using a BD FACSLyric system, and were further analyzed using the FlowJo v10 software (TOMY Digital Biology).
6
Multiparameter Flow Cytometry Analysis
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Cell suspensions were analysed using BD LSRII research flowcytometry, after staining with combinations of the following antibodies and their isotype controls: Anti-CD4-APC-CY7 (BD Pharmingen, USA), anti CD25-Alexa Fluor 700 (BD Pharmingen, USA), anti CCR4-PE-CY7 (BD Pharmingen, USA), anti CCR6-Alexa Fluor 647 (BioLegend), anti CD127-Alexa Fluor 647 (BD Pharmingen, USA), anti FoxP3-Alexa Fluor 488 (BD Pharmingen, USA), anti GATA-3-Alexa Fluor 647 (BD Pharmingen, USA), and anti Tbet-PE (Santa Cruz). For intracellular staining, cells were fixed and permeabilised using the cytofix/Cytoperm kit (BD Biosciences, USA) as per manufacturer's protocol. Data were analysed using FlowJo ® version 7.6 (Tree Star Inc., USA). Lymphocyte population was selected then identification of pure CD4 + proportion of T cells was gated on isotype control. The cell populations were calculated as percentages of CD4 + T cells to the whole lymphocytic count and presented as mean SD. Identification of different T cell subsets and gating is shown in Figure S6, S7, S8 and S9.
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