Cervical tissue from healthy donors was obtained and digested, and the resulting cell suspension was stained for viability with Aqua vital dye (Invitrogen) and the following antibodies: CD69-FiTC (FN50 dilution 1/20, cat. no. 557049), CD45-PE (HI30, dilution 1/100, cat. no. 560975), CD3-PerCP (SK7, dilution 1/10, cat. no. 345766), CD19-V500 (HIB19, dilution 1/50, cat. no. 561121), PD1-BV421 (EH12.1, dilution 1/20, cat. no. 562516) (all from BD Biosciences), CD4-APC (OKT4 BioLegend, dilution 1/80, cat. no. 300514). Aqua−CD19−CD45+ CD3+ CD4+ T cells were acquired in a BD FACSAriaTM II Cell Sorter and separated according to their CD69 expression into CD69+ (TRM) and CD69− (non-TRM). Sorted cells were then infected with 3655 TCID50 of the viral stock HIV-1BaL for 4 h at 37 °C or with medium in control conditions, washed with PBS and cultured in a 96-wells plate. After three days, cells were stained for viability with Live/Dead Fixable Far Red (Invitrogen), CD69-FiTC (FN50 dilution 1/20, cat. no. 557049) and HLA-DR-PerCP-Cy5.5 (G46-6, dilution 1/100, cat. no. 560652), before staining for p24 as described above. Fixed cells were finally acquired in a BD FACS Calibur flow cytometer and analyzed with FlowJo vX.0.7 software (TreeStar).
Cd69 fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD69-FITC is a fluorescently labeled antibody that binds to the CD69 antigen, which is a cell surface glycoprotein expressed on activated T cells, B cells, and natural killer cells. It is commonly used in flow cytometry applications for the detection and analysis of activated immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd62l fitc, by Thermo Fisher Scientific (4 mentions)
Facscanto 2, by BD (4 mentions)
Lsrfortessa, by BD (3 mentions)
Cd25 pe, by Thermo Fisher Scientific (3 mentions)
Cd4 apc, by Thermo Fisher Scientific (3 mentions)
Foxp3 apc, by Thermo Fisher Scientific (3 mentions)
Tbet efluor660, by Thermo Fisher Scientific (3 mentions)
Cd8 percp cy5, by Thermo Fisher Scientific (2 mentions)
Gl 7 fitc, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (2 mentions)
Cd11b pe, by Thermo Fisher Scientific (2 mentions)
Cd4 percp, by BD (2 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions)
Cd44 pe, by Thermo Fisher Scientific (2 mentions)
Ifn γ fitc, by Miltenyi Biotec (2 mentions)
Granzyme b apc, by Thermo Fisher Scientific (2 mentions)
Fixation permeabilization buffer set, by Thermo Fisher Scientific (2 mentions)
Cd4 percp cy5, by Thermo Fisher Scientific (2 mentions)
Cd8a apc cy7, by BD (2 mentions)
Rorγt pe, by Thermo Fisher Scientific (2 mentions)
24 protocols using cd69 fitc
1
Characterizing cervical tissue-resident memory T cells
2
Comprehensive Immune Cell Profiling
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Lung and nasal tissue mononuclear cell suspensions were prepared by mechanical (chopping with a scalpel) followed by enzymatic disruption of tissue for 1 h at 37°C with Collagenase D (1 mg/ml; Sigma-Aldrich) and DNAse I (20 U/ml; Sigma-Aldrich). Next, lungs or spleens were passed through a 40-mm cell strainer to a obtain single-cell suspension, followed by RBC lysis. The cells were incubated with CD16/CD32 FcgRIII (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. To detect cytokines, cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A (5 mg/ml) for 4 h at 37°C. The following surface Abs were used: CD45R-PE, CD3-BV421, CD44-BV605 (Biolegend), CD62L-PE-CF594 (BD), CD103-BV786, CD4-APC-eF780, CD69-FITC (eBioscience). For detection of intracellular cytokines, cells were fixed in 2% PFA and permeabilized with 0.5% saponin (Sigma-Aldrich, Ireland), followed by staining with IL-17A–PerCP-Cy5.5 and IFN-γ-BV650 (eBiosciences). Fluorescence minus one or non-specific isotype Abs were used as controls. Flow cytometric analysis was performed on an LSR Fortessa, and data were acquired using Diva software (BD Biosciences). The results were analyzed using FlowJo software (TreeStar).
3
Multiparameter Tumor Immune Profiling
4
Multiparameter Flow Cytometry Analysis
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Splenocytes, mesenteric lymph node cells, and inguinal lymph node cells were isolated from the indicated mouse and then stained with anti-mouse CD4-PE/Cy7, CD8-Percp/Cy5.5, CD25-PE, CD69-FITC, CD44-APC-Cy7, CD62L-FITC, B220-Percp/Cy5.5, GL-7-FITC, and CD95-PE antibodies (eBioscience, San Diego, CA, USA) for 15 min at 4 °C. For TFH cell analysis, cells were stained with anti-mouse CXCR5-biotin for 30 min at 4 °C followed by anti-mouse CD44-APC/Cy7 and CD4-PE/Cy7 and streptavidin-APC staining. After fixation and permeabilization using a Foxp3 Staining Kit (eBioscience), intracellular Bcl-6 was stained using anti-mouse Bcl-6-PE (BD Bioscience, San Jose, CA, USA) for 40 min at room temperature. Cells were examined using a FACSCanto II system (BD Bioscience), and data were analyzed using FlowJo software (Treestar, Ashland, OR, USA).
5
Quantification of Antigen-Specific T Cells via HLA Tetramers
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PE-conjugated HLA-A*0201 RMSAPSTGGV tetramer (H3.3K27M-tetramer) was produced by the National Institute of Allergy and Infectious Disease tetramer facility within the Emory University Vaccine Center (Atlanta, GA) using the peptide synthesized by A&A Laboratories. PE-conjugated HLA-A*0201/RMSAPSTGGV Dextramer was purchased from Immudex. Cells were stained with tetramer (10 µg/ml) or dextramer in PBS containing 1% BSA for 15 min at 4°C (for tetramer) or room temperature (for dextramer), followed by surface staining for various T cell markers at 4°C. Cells were then washed with PBS containing 0.1% BSA. For some experiments, T cells were stained with tetramer, followed by anti–human CD3 FITC (344803; BioLegend), CD4-PerCPCy.5.5 (317427; BioLegend), CD8 APC (344722; BioLegend), CD69 FITC (11-0699-42; eBioscience), or PD-1-PECy7 (561272; BD Biosciences) along with the suitable isotype control antibodies. Intracellular cytokine staining was performed using Fixation/Permeabilization Solution kit (54714; BD Biosciences) according to the manufacturer’s instructions. T cells were then stained with anti–human Granzyme-B-BV421 (563389; BD Biosciences). The cells were acquired using a Sony SH800 flow cytometer and analyzed using FlowJo software v.10.
6
Immune Profiling of Ophthalmic Blood
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First, 20 μL of peripheral blood was collected from the ophthalmic vein and added into tubes containing 4 μL of 1% heparin sodium. Red blood cell lysis buffer was added (Biosharp, China) and the blood was lysed in the dark for 2 min. Then, 2 mL of PBS was added to wash off the lysate. Next, the cells were redissolved in PBS and stained in the dark for 30 min with CD206-phycoerythrin (PE), CD11b-PE, CD25-Fluorescein isothiocyanate (FITC), CD69-FITC, and CD71-FITC (eBioscience, USA), separately. The expression levels of immune response CD markers were measured by flow cytometry (CytoFLEX S, Beckman Coulter, USA).
7
T Cell Activation Assay with DCs
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The T cells were labelled with 5 μM carboxyfluorescein succinimidyl ester (CFSE; Sigma–Aldrich, USA) according to the manufacturer’s instructions, and then cocultured with DC, Rapa-DC or CsA-DC. After 5 days of coculture, the cells were harvested and labelled with the following fluorochrome-conjugated monoclonal antibodies: CD3-APC (clone: OKT3, eBioscience, USA), CD69-FITC (clone: FN50, eBioscience, USA) and CD25-PE (clone: BC96, BD Pharmingen, USA) to identify T cells or CD11c-APC and PD-L1-FITC (clone: MIH1, BD Pharmingen, USA) to identify DCs. All procedures were performed according to the manufacturers’ protocols. The cells were prepared for flow cytometry analysis and analysed as described above.
8
Multiparametric Flow Cytometry of Murine Splenocytes
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A total of 1 × 106 lymphocytes were acquired and collected from mice spleen, and then 0.5 μg anti-mouse CD16/32 monoclonal antibody (Biolegend, USA) and 5 μL rat serum were used to block the Fc receptor for 10 min. For measuring the number of total CD4+ T cell and naïve CD4+ T cell, splenocytes were incubated with 1 μg CD3-APC-Cy7 (Biolegend), CD4-APC (eBioscience, Thermo Fisher Scientific, USA), TCRβ-PE and CD62L-FITC (eBioscience, Thermo) for 30 min. For detecting cell activation, cells were stained with 1 μg CD25-PE and CD69-FITC (eBioscience, Thermo). For evaluating cell surface protein expression, EGFR (Thermo)-PE (eBioscience) and Glut1 (Proteintech, China) -FITC (eBioscience) were incubated for 30 min. To measure cell death, splenic cells were stained with PI and Annexin V (BD Biosciences, San Jose, CA, USA) for 15 min at room temperature. Cells were analyzed by FACSCanto II cytometer (BD Biosciences, San Jose, CA, USA). All data were analyzed by Flowjo10 software (Tree Star, Ashland, OR, USA).
9
Multiparametric Flow Cytometry Analysis
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Surface staining was performed in PBS with 1% BSA using the indicated antibodies – CD25-APC, CD69-FITC (eBioscience), CD4-PerCP (BD Pharmingen), Anti-mouse DO.11.10-TCR Biotin (Invitrogen), Streptavidin PerCP (BD Pharmingen). Intra-cellular staining was performed for detection of intra-cellular Notch1, T-bet, GATA3, and FoxP3 using the FoxP3 staining buffer set (eBioscience) and the following antibodies: anti-Human/Mouse Notch1-PE, anti-Human/Mouse T-bet PE-Cy7, anti-Human/Mouse GATA3 eFluor 660, anti-Mouse/Rat FoxP3 Alexa 488 (eBioscience). For detection of intra-cellular cytokines, cells were harvested at indicated time points and re-stimulated with plate-bound anti-CD3ε in the presence of Golgi Plug (IFN-γ) or Golgi Stop (IL-4) (BD Pharmingen) for 5 h. Intra-cellular cytokine staining was performed using the BD Cytofix/CytoPerm plus kit and cytokines detected using anti-Mouse IFN-γ FITC and anti-Mouse IL-4 PE (BD Pharmingen). Flow cytometry data was acquired on a FACS LSR II (BD) and analyzed using FlowJo software (Trestar) after gating on CD4+ T cells or as indicated.
10
Evaluating Iberodomide and JQ1 Effects on T Cell Activation and Apoptosis
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Primary CD4+ T cells isolated from buffy coats of healthy volunteers were treated with 10 μM of iberdomide, 500 nM of JQ1, or both compounds, with PMA/ionomycin used as a positive control. The cells were examined by flow-cytometry at 24 and 48 h, live cells were gated using forward scatter area versus side scatter area profiles (FSC-A, SSC-A). Cells were then stained for expression of Annexin V to examine the percentage of cells undergoing apoptosis and with the surface receptor CD69 to measure T cell activation. For Annexin V and CD69 staining, 106 cells were washed with PBS supplemented with 3% FBS and 2.5 mM CaCl2 followed by staining with Annexin V-PE (Becton and Dickinson), CD69-FITC (eBIOSCIENCE) for 20 min at 4C in the presence of 2.5 mM CaCl2. Cells were then washed with PBS/FBS/CaCl2 and analyzed by flow cytometry. Between 2–4 × 105 events were collected per sample within 3 h after staining on an LSRFortessa (BD Biosciences, 4 lasers, 18 parameters) and analyzed using FlowJo software (version 9.7.4, Tree Star).
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