Activated Tfh cells in the spleen were labeled with FVS-440UV, CD45-BV786, CD3-FITC (Clone 145-2C11, BD), CD4-V500, CD8-BV605, and CXCR5-PE (Clone 2G8, BD) following surface antigen staining procedure. Subsequently, cell pallets were suspended in 1 ml fixation buffer (Cat. 554655, BD) for 15 min under room temperature and washed with 1 ml perm/wash buffer (Cat. 557885, BD) twice. Anti-mouse Bcl-6-Alexa Fluor 647 (Clone K112-91, BD) was added at 1 : 50 ratio and incubated in the dark for 30 min at 4°C. Cells were then washed twice and resuspended in cold PBS for flow cytometry assay. Plasma cells in peripheral blood cells (PBMCs) were labeled with FVS-440UV, CD45-BV786, CD3-FITC, B220-PE-Cy7, CD19-APC-cy7 (Clone 1D3, BD), and CD138-BV421 (Clone 281-2, BD) following the procedure described earlier.
Cxcr5 pe
Manufactured by BD
Sourced in United States
CXCR5-PE is a flow cytometry reagent that binds to the CXCR5 receptor. It can be used for the identification and analysis of CXCR5-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Fixation buffer, by BD (1 mentions)
Cd138 bv421, by BD (1 mentions)
Cd4 v500, by BD (1 mentions)
Cd19 apc cy7, by BD (1 mentions)
Fvs 440uv, by BD (1 mentions)
Cd45 bv786, by BD (1 mentions)
Cd8 bv605, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cd3 fitc, by BD (1 mentions)
B220 pe cy7, by BD (1 mentions)
Cd27 fitc, by BD (1 mentions)
Cxcr3 apc, by BD (1 mentions)
Ifnγ pe antibodies, by BD (1 mentions)
Ccr6 pe, by BD (1 mentions)
Cd40l pe, by BD (1 mentions)
Cd4 fitc, by BD (1 mentions)
Cd38 pe, by BD (1 mentions)
Streptavidin bv421, by BD (1 mentions)
Cd4 bv605, by BD (1 mentions)
Cxcr5 biotin, by BD (1 mentions)
5 protocols using cxcr5 pe
1
Activated Tfh Cells and Plasma Cells Profiling
2
Multiparameter flow cytometry for cTfh subsets
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PBMCs were stained with CD4-FITC, CXCR5-PE, CXCR3-APC, CCR6-PE, CD27-FITC, CD38-PE, CD40L-PE, and IFNγ-PE antibodies (BDPharmingenTM, USA) and incubated at 4°C for 30 min in the dark. After the antigen–antibody incubation was completed, the specimen was cleaned 3 times with 2.5 mL of flowing washing solution (Hyclone, USA)/PBS, then resuspended in 500 μL of PBS, and put in storage at 4°C for performing upper flow cytometry analysis. The isotype control antibody was used to adjust the compensation of each channel and set the gate parameters. FlowJo software (Version 7.6.1, Tree Star Inc., USA) was employed to analyze the data obtained by flow cytometry. CD4+CXCR5+CXCR3+CCR6 indicates Th1-like cTfh cells, CD4+CXCR5+CXCR3-CCR6 indicates Th2-like cTfh cells, and CD4+CXCR5+CXCR3-CCR6+ indicates Th17-like cTfh cells.
3
Multiparameter Immunophenotyping of Influenza-Infected Ferret Lymph Nodes
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Lymph node cell suspensions from influenza infected ferret were stained with the following panel: live/dead Blue (Thermo Fisher), CD79a PerCP-Cy5.5 (HM47; BioLegend)24 (link), CD8 AF700 (OKT8; Thermo)24 (link), CD4 FITC (from CSIRO)25 (link), BCL6 AF647 (K112-91; BD), BCL6 AF647 (IG191E/A8; BioLegend), CXCR5 BV421 (L138D7; BioLegend), CXCR5 BB515 (RF8B2; BD), CXCR5 PE (2G8, BD); CXCR5 Biotin (in-house), Streptavidin BV421 (BD), PD-1 BV786 (29F.1A12; BioLegend), PD-1 BV421 (EH12.2H7; BioLegend), PD-1 PE (in-house). For BCL6 staining, cells were fixed, permeabilized, and stained using the BD Transcription Factor Buffer kit (BD) according to the manufacturer’s instructions. Macaque LN suspensions were stained with Live/dead Aqua (Thermo Fisher), CD4 BV605 (L200; BD), CXCR5 PECy7 (MU5UBEE, Thermo Fisher), and CD3 BUV737 (SP34-2, BD). All samples were acquired on a BD LSR Fortessa using BD FACS Diva and data was analyzed in FlowJo v10.
4
Multicolor Flow Cytometry Antibody Panel
5
Multiparametric Immune Cell Profiling
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To examine the expression of surface markers and intracellular molecules, cells were incubated with FcR blocking reagent (Miltenyi, Germany) for 10 min followed by primary antibodies on ice in the dark for 30 min. The antibodies used for surface marker analysis included anti-human CD4-FITC, CXCR5-PE, PD-1-PE-cy7, BCL-6-APC, CD25-PE, CD19-FITC, CD138-PE (BD Pharmingen, USA), and anti-mouse CD4-FITC, CXCR5-APC, PD-1-PE, and CD138-PE (eBioscience, USA). For intracellular staining, cells were cytofixed and cytopermed using the CytofixCytoperm Plus kit (BD Pharmingen, USA) and stained with intracellular antibodies, including anti-human IL-17-APC, IFN-γ-PE, IL-4-PE-cy5, Foxp3-APC (BD Pharmingen, USA), 5-mC and 5-hmC (Abcam, USA) for an additional 30 min on ice in the dark. For some experiments, apoptosis was detected using a FITC Annexin V Apoptosis Detection Kit II (BD Pharmingen, USA). Data were acquired by flow cytometry (BD, Canto II, USA) and analyzed using FlowJo (Tree Star, USA).
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