The following conjugated monoclonal antibodies from eBioscience/ThermoFisher were used: CD3 APC-efluor780 (17A2), CD4 efluor450 (RM4-5), CD45.2 APC (104), CD62L PE-Cy7 (MEL-14), CD69 PE (H1.2F3), Foxp3 APC (FJK-16s), IL-17A PE-Cy7 (eBio17B7), IFN-gamma efluor660 (XMG1.2), ROR-gamma-t PE (B2D), and T-bet efluor660 (eBio4B10). Dead cells were excluded by LIVE/DEAD® Fixable Dead Cell Stain Kit (Life Technologies). For intracellular cytokine staining, cells were stimulated with Phorbol 12-myristate 13-acetate (0.1 μg/ml/1; Sigma-Aldrich) and ionomycin (1 μg/ml/1; Sigma-Aldrich) for 4h, the last 2h in the presence of Brefeldin A (5 μg/ml), stained for surface markers, fixed using Foxp3/Transcription Factor Fixation/Permeabilization Kit (Affymetrix/eBioscience) according to manufacturer’s instruction, and stained with the respective antibodies against cytokines or transcription factors diluted in PBS containing 0.25% BSA and 0.5% of Saponin. The acquisition was performed on an LSR II flow cytometer (Becton Dickinson), and data were analyzed with FlowJo software (Tree Star, Inc.). Single stains were used for compensation and fluorescence minus one (FMO) controls for gating. For the MT-CO1 (Abcam, EPR19628), staining cells were fixed with ice-cold methanol.
Cd69 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD69-PE is a fluorescent-labeled antibody used in flow cytometry applications to detect and quantify the CD69 protein, which is a marker of early T-cell activation. The PE (phycoerythrin) fluorescent dye is conjugated to the CD69 antibody, allowing for the identification and analysis of CD69-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (4 mentions)
Cd4 efluor450, by Thermo Fisher Scientific (2 mentions)
Mouse fc block, by BD (2 mentions)
Cd25 alexafluor488, by Thermo Fisher Scientific (2 mentions)
Cd27 pe, by Thermo Fisher Scientific (2 mentions)
Cd19 apc, by Thermo Fisher Scientific (2 mentions)
Cd8 fitc, by Thermo Fisher Scientific (2 mentions)
Cd3 apc, by Thermo Fisher Scientific (2 mentions)
Cd44 apc, by Thermo Fisher Scientific (2 mentions)
Cd8 apc, by Thermo Fisher Scientific (2 mentions)
Cd4 fitc, by Thermo Fisher Scientific (2 mentions)
Cd8 pe cy7, by Thermo Fisher Scientific (2 mentions)
Cd3 fitc, by Thermo Fisher Scientific (2 mentions)
Cd3 pe cy7, by Thermo Fisher Scientific (2 mentions)
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cd3 apc efluor780, by Thermo Fisher Scientific (1 mentions)
Cd62l pe cy7, by Thermo Fisher Scientific (1 mentions)
Foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
17 protocols using cd69 pe
1
Comprehensive T Cell Immunophenotyping
2
Multiparametric Immunophenotyping of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleen and MLN single cell suspensions (0.5–1 × 106 cells/well) were incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block, BD Biosciences, San Jose, CA, USA) in PBS supplemented with 1% BSA and 5% FCS for 15 min on ice to block non-specific binding sites. Subsequently, cells were incubated for 30 min with the following antibodies: CD4-PerCP Cy5.5, CCR6-PE (both from Biolegend, San Diego, CA, USA) CD8a-PECy7, CD69-PE, CD25-Alexa Fluor 488, CD3-PerCP Cy5.5, CD27-PE, CD19-APC and B220-FITC (all from Thermo Fisher). For intracellular staining, cells were first fixated and permeabilized with Foxp3 Staining buffer set (Thermo Fisher) according to manufacturer’s protocol, followed by incubation with Foxp3-PECy7 (Thermo Fisher), RORγT-Alexa Fluor 647 (BD) or Tbet-eFluor 660 (Biolegend). Dead cells were excluded using Fixable Viability Dye eFluor® 780 (Thermo Fisher). Stained cells were measured by FACS Canto II (BD Biosciences) and analyzed using Flowlogic software version 7 (Inivai Technologies, Mentone, VIC, Australia).
3
Plasmid-based PTEN Gene Therapy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The three plasmid systems, including target gene plasmid (pAAV-CAG-mCherry), capsid plasmid (pAAV-6, pAAV-1, pAAV-7m8, pAAV-7, pAAV-8, pAAV-10) and helper plasmid (pHelper), were all purchased from Fenghui Biotechnology (Hunan, China). The complementary DNA (cDNA) encoding the mouse PTEN gene was synthesized from GENEWIZ (Suzhou, China). pAAV-CAG-PTEN was obtained by replacing mCherry gene in pAAV-CAG-mCherry with PTEN gene. Reverse the PTEN gene sequence in pAAV-CAG-PTEN to get pAAV-CAG-PTENR.
The anti-PD-1 used in this study is AUNP-12, a polypeptide purchased from Selleck (USA) with a purity of 99.20%. Cy5-AUNP-12 was purchased from Guoping pharmaceutical co (Anhui, China) with a purity of 90%. CpG was synthesized in GENEWIZ (Suzhou, China) with 90% purity.
Antibodies for Western blot (WB) and Immunofluorescence (IF): anti-mouse PTEN, CRT, β-actin and anti-rabbit FITC, CY3 fluorescent secondary antibodies were purchased from ABclonal Tech (Wuhan, China). Antibodies for flow cytometry test: CD11c-PE, CD80-FITC, CD86-APC, CD3-APC, CD8-FITC, CD4-APC-eflour780, CD69-PE, CD274-PE, CD11b-FITC, CD206-PE-Cy7, CD86-APC, IL-4-PE, IFN-γ-APC, TNF-α-eflour450, IL-2-PE5.5, CD4-APC-eflour780, CD44-APC, CD62L-PE were all purchased from Invitrogen (USA).
The anti-PD-1 used in this study is AUNP-12, a polypeptide purchased from Selleck (USA) with a purity of 99.20%. Cy5-AUNP-12 was purchased from Guoping pharmaceutical co (Anhui, China) with a purity of 90%. CpG was synthesized in GENEWIZ (Suzhou, China) with 90% purity.
Antibodies for Western blot (WB) and Immunofluorescence (IF): anti-mouse PTEN, CRT, β-actin and anti-rabbit FITC, CY3 fluorescent secondary antibodies were purchased from ABclonal Tech (Wuhan, China). Antibodies for flow cytometry test: CD11c-PE, CD80-FITC, CD86-APC, CD3-APC, CD8-FITC, CD4-APC-eflour780, CD69-PE, CD274-PE, CD11b-FITC, CD206-PE-Cy7, CD86-APC, IL-4-PE, IFN-γ-APC, TNF-α-eflour450, IL-2-PE5.5, CD4-APC-eflour780, CD44-APC, CD62L-PE were all purchased from Invitrogen (USA).
4
Quantifying IFN-γ and TCR Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IFN‐γ production by TCR‐transduced primary T cells was quantified using standard enzyme‐linked immunosorbent assays (ELISA) according to the manufacturer's instructions (Diaclone, Besançon, France). Responder T cells were co‐cultured with stimulator cells at a ratio of 1:5 (responder: stimulator) for 16 h at 37°C in TCM using 25 IU/mL IL‐2 instead of 100 IU/mL IL‐2. To measure activation of TCR‐transduced Jurkat E6∆TCR cell‐lines upregulation of activation marker CD69 was analyzed using flow cytometry. Responder TCR‐transduced Jurkat E6∆TCR cells were stimulated with HLA‐A*02:01‐transduced K562 cell‐lines with or without exogenous peptide loading (10−6 M) at a ratio of 1:10 (responder:stimulator, R:S) in stimulator medium for 16 h at 37°C. After O/N incubation, cells were washed twice before adding CD69‐PE (Invitrogen), mTCR‐Cβ‐APC (BD), and CD8‐PerCP (BD) monoclonal antibodies for 30 min at 4°C. All analyses were performed on a FACS Calibur (BD), and analyzed using Flowjo Software (TreeStar, Ashland, USA).
5
Phenotypic Identification of Migratory DCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly isolated mesenteric lymph nodes (MLNs) were analyzed by flow cytometry (14 (link)). Identification of migratory DCs in the MLNs was performed by gating CD103+ cells out of CD11c+ MHC-II cells in the MLNs (the gating strategy is shown in Figure 2 A
6
Jurkat Cell Surface Marker Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell surface marker staining, Jurkat cells were washed twice with PBS/0.5% BSA, and incubated with 50 μl of the antibody diluted in PBS/0.5% BSA for 30 min in the dark. Antibodies used include CD69-PE (12–0699, eBioscience, San Diego, CA) or Mouse IgG1 K isotype control PE (12–4714, eBioscience) and CD4-APC (MHCD0405, Caltag, Buckingham, UK). Cells were washed twice with PBS and then resuspended in 200 μl PBS/2.5% formalin, washed twice with PBS and twice with PBS/0.5% BSA. Fixed cells were subjected to flow cytometry analysis on a FACS Calibur in the Flow Cytometry Core Facility in UT Southwestern Medical Center. Data analysis was performed using FlowJo version 9.6.1.
7
Quantifying CD69 Expression in Jurkat Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Jurkat cells (1 × 105) were stimulated with P/I (10/80 ng/mL) for 24 h. The cells were then stained with CD69-PE (12-0699, 1:50, eBioscience, San Diego, CA, USA) in staining buffer (PBS with 1% FBS) on ice for 20 min and then washed three times with staining buffer. The CD69-positive population was evaluated by a flow cytometer (FACSCalibur; BD Biosciences). Data were analyzed with FlowJo software (BD Biosciences). The signal obtained for the cells treated without or with GTE alone and stained with mouse IgG1 isotype control-PE (12-4714, 1:50, eBioscience) was used as a control respectively.
8
Flow Cytometric Immunophenotyping and ATP Detection
9
Immune Cell Profiling in Murine Spleen
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
10
Multiparameter Flow Cytometry Analysis
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!