T cells were washed in staining buffer (SB) consisting of phosphate-buffered saline (PBS) containing 0.1% human serum albumin (HSA) and 0.1% sodium azide before staining for 20 min at RT. The cells were then washed in SB and fixed in SB containing 1% paraformaldehyd. For intracellular staining, T cells were stimulated for 6 h or overnight with APCs, loaded or not with p573, at a T-cell to target ratio of 1:2 and in the presence of BD GolgiPlug and BD Golgistop at a 1/1,000 dilution. Cells were stained both extracellular and intracellular using the PerFix-nc kit according to the manufacturer's instructions (Beckman Coulter Inc., USA). The following antibodies were used: Vβ3- FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor450, CD4-eFluor 450, CD4-PE-Cy7, CD8-APC, CD8-eFluor 450, CD8-PE-Cy7, CD56-PE-Cy5.5 (BD Biosciences, USA) and CD107a-PE-Cy5 (BD Biosciences, USA), CXCR2-PE, IFNγ-FITC, IL-2-APC, TNF-α-PE (BD Biosciences, USA), CD261/TRAIL-R4-PE (BD Biosciences, USA). MART-1 (aa 26–35)-specific TCR was detected with dextramer staining (Immudex) following the manufacturer's recommendations. All antibodies were purchased from eBioscience, except where noted. Cells were acquired on a BD LSR II flow cytometer and the data analyzed using FlowJo software (Treestar Inc.).
Cd4 efluor 450
Manufactured by BD
Sourced in France, United States
The CD4-eFluor 450 is a fluorescently-labeled antibody that binds to the CD4 surface protein on T cells. It is used in flow cytometry applications to identify and quantify CD4+ T cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace violet ctv, by Thermo Fisher Scientific (2 mentions)
Annexin 5 pacific blue, by BioLegend (2 mentions)
Cell fixation permeabilization kit reagents, by BD (2 mentions)
Lsrii platform, by BD (2 mentions)
Cd4 pe, by BD (2 mentions)
Cd4 apc, by BD (2 mentions)
Ifn γ fitc, by BD (1 mentions)
Tnf α pe, by BD (1 mentions)
Cd107a pe cy5, by BD (1 mentions)
Il 2 apc, by BD (1 mentions)
Perfix nc kit, by Beckman Coulter (1 mentions)
Cd8 efluor 450, by BD (1 mentions)
Cxcr2 pe, by BD (1 mentions)
Cd8 pe cy7, by BD (1 mentions)
Cd4 pe cy7, by BD (1 mentions)
Cd8 apc, by BD (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Dextramer siinfekl h 2kb pe, by Immudex (1 mentions)
Cd8 percp, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 protocols using cd4 efluor 450
1
Multiparametric T cell Characterization
2
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were re-suspended in FACS buffer (PBS with 1% BSA, 2 mM EDTA) and incubated with Fc blocker for 10 min on ice. After washing with FACS buffer, cells were incubated with the desired antibodies at optimal concentrations for 20 min on ice in the dark. Cells were then washed with FACS buffer twice and re-suspended in 200 µL FACS buffer for flow cytometry analysis (LSRII, BD Biosciences, NJ, USA). For intracellular staining, cells were stained with surface markers as described above, treated with cell fixation/permeabilization kit reagents (BD Biosciences, NJ, USA) and then incubated with antibodies for desired intracellular markers. CD4-PE, CD4-APC, or CD4-eFluor 450 was purchased from BD Biosciences. Tfh cell staining was performed using a three-step staining protocol.24 (link) Annexin V-pacific Blue (Biolegend, CA, USA) was used to assess the apoptotic status of cells. CellTrace Violet (CTV) (Thermo Fisher Scientific, MA, USA) was applied to identify cell proliferation status, and the proliferation index (P.I.) at each division peak was calculated following the manufacturer’s instructions. Flow cytometry analysis was performed using a LSRII platform (BD Biosciences, NJ, USA) and data were analyzed using FlowJo software 7.6 (TreeStar, CA, USA).
3
CFSE-Labeled T Cell Proliferation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A single-cell suspension was prepared from the spleens and lymph nodes of transgenic OT-I and OT-II mice (Ghent University, Ghent, Belgium). T cells were isolated using a CD8a+ and CD4+ T cell isolation kit for mouse and an AutoMACS Pro Separator (Miltenyi Biotec, Germany). Subsequently, the isolated T cells were labeled with (carboxyfluorescein diacetate succinimidyl ester (CFSE); Thermo Fisher Scientific) by incubating 50×106 cells/mL PBS with 5 µM of CFSE for 7 min at 37°C. The reaction was blocked by adding ice-cold PBS +10% FBS (Life Technologies). A total of 2×106 OT-I or OT-II cells were injected into the tail vein of C57BL/6 recipient mice. The mice were treated 2 days later with plasmid injection and electroporation to the ear pinna. Mice were sacrificed 4 days later to collect the draining lymph nodes for single-cell suspension preparation. Flow cytometric measurement was performed after staining the cells with Aqua Live/Dead (Thermo Fisher), CD19 APC-Cy7, CD8 PerCP (for OT-I proliferation), CD4 eFluor 450 and Valpha2 TCR PE (for OT-II proliferation) and Fc block (all BD Biosciences, USA). Dextramer SIINFEKL H-2kb PE (Immudex, Denmark) was added for OT-I proliferation measurement. The read out was performed using a BD FACSVerse (BD Biosciences, USA).
4
Multiparameter Flow Cytometry Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were re-suspended in FACS buffer (PBS with 1% BSA, 2 mM EDTA) and incubated with Fc blocker for 10 min on ice. After washing with FACS buffer, cells were incubated with the desired antibodies at optimal concentrations for 20 min on ice in the dark. Cells were then washed with FACS buffer twice and re-suspended in 200 µL FACS buffer for flow cytometry analysis (LSRII, BD Biosciences, NJ, USA). For intracellular staining, cells were stained with surface markers as described above, treated with cell fixation/permeabilization kit reagents (BD Biosciences, NJ, USA) and then incubated with antibodies for desired intracellular markers. CD4-PE, CD4-APC, or CD4-eFluor 450 was purchased from BD Biosciences. Tfh cell staining was performed using a three-step staining protocol12 (link). Annexin V-pacific Blue (Biolegend, CA, USA) was used to assess the apoptotic status of cells. CellTrace Violet (CTV) (Thermo Fisher Scientific, MA, USA) was applied to identify cell proliferation status, and the proliferation index was calculated following the manufacturer’s instructions. Cell cycle status was assessed by 7-AAD (Tonbo Biosciences, CA, USA) and BrdU (Biolegend, CA, USA) staining. Flow cytometry analysis was performed using a LSRII platform (BD Biosciences, NJ, USA) and data were analyzed using FlowJo software 7.6 (TreeStar, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!