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8 protocols using cd8a apc cy7

1

Lung Immune Response to RBD Vaccination

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Left lungs from HD-Ad_RBD vaccinated and control mice were harvested and digested for 45 min at 37 °C in digestion buffer containing liberase 2 μg/ml and Type IV DNase I 25 units/ml. The lung infiltrated cells were cultured with purified RBD protein at 10 μg/ml for 12 h at 37 °C followed by a 6 h treatment with GolgiPlug (BD 555028). After blocking with FcγIII and FcγII receptors antibodies (BD Pharmingen, 553142), cells were stained with live/dead fixable cell stain (Invitrogen 34955), CD44 BV510, CD4 BV711, and CD8a APC-Cy™7 (BD) antibodies. Stained cells were fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization (BD 555028), and then intracellularly stained with anti-IFN-γ APC (BD). Cells were analysed on a Becton Dickinson LSR II CFI (SickKids Flow Cytometry Facility), using Flowjo × 10.0 software.
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2

Multiparameter Flow Cytometry for Immune Response

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Multiparameter flow cytometry was performed in accordance to BD Pharmingen™ Protocol. In brief, splenocytes were harvested at day 14 post second boost; 5 × 106 cells were stimulated in a Nunc™ 96-well conical-bottom plate (Thermo Scientific™, USA) for 6 h at 37 °C with 10 μg of M2eh peptide and 1 μg of influenza virus A/Aichi/2/68 (H3N2) in the presence of 1 μg/ml of Brefeldin A (BD Bioscience, USA) and purified hamster anti-mouse CD28. The cells were then washed, and Fc receptors were blocked using CD16/CD32 antibodies (Mouse BD Fc Block, BD Pharmingen, USA) for 30 min. Next, cells were incubated with Zombie Aqua (Zombie Aqua™ Fixable Viability Kit, Biolegend, USA) to enable gating of live cells during analysis and subsequently stained with antibodies (CD3e-FITC, CD8a-APC-Cy7, CD4-PerCP, CD62L-PE-Cy7, CD44-APC) at 4 °C for 30 min (antibodies from BD Pharmingen, USA). Cells were permeabilized using the BD Cytofix/Cytoperm Plus (BD Bioscience, USA) protocol and stained with anti-TNF-α-BV421 or anti-IFN-γ-PE (BD Pharmingen, USA). Sample acquisition (50,000 live CD3+ were collected) was performed with a BD FACS Canto II flow cytometer (Becton Dickinson, USA) and analyzed using Kaluza, version 1.5 (Beckman Coulter, USA).
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3

Multifunctional T-cell Responses to Influenza

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Multi-parameter flow cytometry was performed in accordance with the BD PharmingenTM protocol. In brief, mouse lungs were harvested at day 14 post second boost; lung lymphocytes (5x106) were stimulated in NuncTM 96-well conical bottom plates (Thermo Scientific™, USA) for 6 h at 37°C with 10 μg of M2eh peptide, in combination with 1 μg of either the A/Aichi/2/68 (H3N2) or the A/California/07/09 (H1N1pdm09) influenza virus, in the presence of 1 μg/ml of Brefeldin A (BD Bioscience, USA) and purified hamster anti-mouse CD28. The cells were then washed, and Fc receptors were blocked using CD16/CD32 antibodies (Mouse BD Fc Block, BD Pharmingen, USA) for 30 min. Next, cells were incubated with Zombie Aqua (Zombie Aqua™ Fixable Viability Kit, Biolegend, USA) to enable gating of live cells during analysis, and they were subsequently stained with CD3e-FITC, CD8a- APC-Cy™7, CD4- PerCP, CD62L-PE-Cy™7, or CD44-APC (BD Pharmingen, USA) antibodies at 40 C for 30 min. Cells were permeabilized using the BD Cytofix/Cytoperm Plus (BD Bioscience, USA) protocols and stained with anti-TNF-α-BV421 or anti-IFN-γ-PE antibodies (BD Pharmingen, USA). Sample acquisition (50,000 live CD3+ T-cells were collected) was performed with a BD FACS Canto II flow cytometer (Becton Dickinson, USA) and analyzed using Kaluza version 1.5 (Beckman Coulter, USA).
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4

Immune Cell Profiling in Mice

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Ten-week-old WT and KO mice were weighed and anesthetized by intraperitoneal injection of pentobarbital sodium. Blood was collected from the internal canthus vein, EDTA anticoagulation. The blood count was used to determine the peripheral blood count and perform classification. Immune cells from peripheral blood were incubated with labeled antibodies. The NK1.1-FITC fluorescent-labeled antibody was purchased from Biolegend; CD11b-APC was purchased from Invitrogen; B220–PerCP, F4/80–PE, CD4-PE-Cy, and CD8a-APC-Cy7 were purchased from BD; B220–PerCP-Cy5.5 and CD3-APC were purchased from Biolegend Division; EDTA was purchased from Sigma; and Erythrocyte lysate was purchased from Invitrogen. We then added the cells to TruCOUNT tubes (BD Biosciences) and counted them on an LSR II flow cytometer (Becton Dickinson), before analyzing with FlowJo software (version 10).
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5

Characterization of Immune Cell Populations

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MLNs were removed, placed on ice in Roswell Park Memorial Institute 1640/penicillin-streptomysin 1%, processed through a 70-nm filter to form single-cell suspension, and blocked for 30 min in PBS containing 1% BSA and 5% heat-inactivated FBS. 1x10E6 Cells were incubated at 4ºC for 30 min with CD8a-APC-Cy7; CD11c-PerCP-Cy5.5 and CD25-Pe-Cy7 were used from BD Biosciences and CD4-PerCP-Cy5.5, CD103-APC, and Foxp3-APC from eBiosciences or matching isotype controls. Extracellular stained cells were fixed with the use of 2% paraformaldehyde, and intracellular staining was performed according to the manufacturer's instructions (eBiosciences). The flow cytometry data are shown as a percentage of the control data (Sham).
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6

Comprehensive Immunophenotyping of Murine Cells

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Single cell suspensions from thymus (if large enough), spleen, and bone marrow (from sacrificed animals) were prepared and cells were counted using a hemacytometer or automated cell counter. A subset of cells (approximately 1 x 10 6 ) was used for flow cytometric analyses. In addition, peripheral blood samples were also analyzed by Flow Cytometry. Surface markers used for immunophenotyping included CD45.1, CD45.2, CD 11b, CD8, CD3, CD4, B220 and Gr-I (Antibodies used: CD45.1-APC, CD45.2-PE, CD 11b-PECy7, CD3-FITC and B220-APC-Cy7, CD4-PE-Cy7, CD8a-APC-Cy7, and Gr-1-APC-cy7all from BD Biosciences, San Jose, CA; cat.no. 561873, 560695, 552850, 555274, 552094, 563933, 557654, and 560600, respectively).
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7

Lung T cell subset analysis

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To determine T cell subsets in the lung, freshly isolated cells were blocked for 2 min at 4˚C using anti-mouse CD16/CD32 antibodies (Mouse BD Fc Block; BD Pharmingen, San Jose, CA, USA) and resuspended in FACS buffer (1% bovine serum albumin (BSA) in PBS). Subsequently, cells were washed and stained using combination of different antibodies against CD4-PerCP Cy5 (cat no. 45-0042, clone RM4-5), CD69-FITC (cat no. 11-0691, clone H1.2F3), GATA3-PE (cat no. 12-9966, clone TWAJ), Tbet-eFLUOR660 (cat no. 50-5825, clone eBio4D10), RORγt-PE (cat no. 12-6988, clone AFKJs-9), Foxp3-APC cat no: 17-5773, clone FJK-16s (eBioscience, San Diego, USA), CD8a-APC Cy7 (cat no. 557654, clone 53-6.7) and CD25-FITC (cat no. 553071, clone 7D4CD25 FITC) (BD, Breda, The Netherlands) for 60 min at room temperature. Viable cells were determined by a fixable viability Dye-eFluor® 780 (eBioscience). For intracellular staining, cells were fixed and permeabilized using a fixation/permeabilization buffer set, according to manufacturer’s protocol (eBioscience). Flow cytometric acquisition was conducted with FACS Canto II (BD Biosciences) and results were analyzed using Flowlogic Software (Inivai Technologies, Victoria, Australia).
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8

Multiparameter Flow Cytometry Immunophenotyping

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Aspecific background was blocked using PBS blocking buffer containing 1% BSA and 5% FCS for 30 min. 5 × 105 cells were plated per well and incubated at 4°C for 30 min with different antibodies against CD4-PerCP Cy5 (cat no. 45-0042, clone RM4-5), CD69-FITC (cat no. 11-0691, clone H1.2F3), GATA3-PE (cat no. 12-9966, clone TWAJ), Tbet-eFLUOR660 (cat no. 50-5825, clone eBio4D10), RORγt-PE (cat no. 12-6988, clone AFKJs-9), Foxp3-APC cat no: 17-5773, clone FJK-16s (eBioscience, San Diego, USA), CD8a-APC Cy7 (cat no. 557654, clone 53-6.7) and CD25-FITC (cat no. 553071, clone 7D4CD25 FITC) (BD, Breda, The Netherlands) and matching isotype controls were used. Cells were permeabilized for intracellular staining using fixation/ permeabilization buffer set, according to manufacturer's protocol (eBioscience). Flow cytometry was conducted using FACS Canto II (BD) and analyzed using Flowlogic Software (Inivai Technologies, Victoria, Australia) (32 (link)).
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