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4 protocols using anti cd127 percp cy5

1

Multiparametric Flow Cytometry Analysis

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Cells were labelled with flow antibodies for 15 minutes in the dark in PBS containing 2% BSA. Labelled cells were washed twice and resuspended in PBS containing 2% BSA. The prepared samples were analyzed using an LSR-II flow cytometer (BD Bioscience) or sorted using an Aria II cell sorter (BD Bioscience). Anti-CD19-PeCy7 (561739), anti-CD3-PeCy7 (552774), anti-NK1.1-BV421 (562921), anti-NKp46-FITC (560756), anti-NKp46-AF647 (560755), anti-CD49b-PE (553858), anti-CD49a-PerCP-Cy5.5 (564862), anti-CD62L-APC (553152), anti-T-bet-APC (561264), anti-CD45.2-AF700 (560693), anti-CD45.2-FITC (553772), anti-Gata3-AF647 (560068), anti-RORγt-PE (562607), anti-CD127-V450 (561205), anti-CD117-PE (553869), anti-LPAM-1-APC (562376), anti-Flt3-BV421 (566292), anti-Ly-6A/E-APC (565355), and anti-CD122-PE (553362) were purchased from BD Bioscience. Anti-IFN-γ-AF-700 (505823) and anti-CD25-Pacific Blue (102022) were purchased from Biolegend. Anti-CD253-APC (17-5951-82), anti-Eomes-PE (12-4875-82), and anti-CD127-PerCP-Cy5.5 (45-1271-80) were purchased from eBioscience.
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2

Multi-Marker Immunophenotyping of T Cells

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Anti-CD3-AlexaFluor®700 (UCHT1), anti-CD4-APC-eFluor®480 (RPAT4), anti-CD69-biotin (FN50), anti-CD127-PerCP-Cy5.5 (eBioRDRS), anti-CD27-APC-eFluor®780 (LG.7F9), anti-CD4-FITC (RPA-T4), anti-CD27-PE-Cy7 (LG.7F9), anti-CD45-APC (2D1), and anti-EpCAM-PE (IB7), anti-FoxP3-PE-Cy7 (PCH101), anti-IL-2-PE-Cy7 (MQ1-17H12), anti-IFNγ-APC-eFluor®780 (4S.B3), anti-TNFα-PerCP-Cy5.5 (Mab11), anti-PD-1-APC (MIH4), anti-PD-L1-PE-Cy7 (MIH1), mIgG1κ-PE-Cy7 (P3.6.2.8.1), anti-CD45RAFITC (HI100), anti-CD45RO-biotin (UCHL1), CD62L-PE-Cy7 (DREG-56), CCR7-APC-efl780 (3D12), anti-CD3 (OKT3), and anti-CD28 (CD28.2) were purchased from eBioscience (San Diego, CA). Anti-CD8-BV510 (RPA-T8) was purchased from BioLegend (San Diego, CA). Anti-CTLA-4-Biotin and anti-Ki67-PerCP-Cy5.5 (B56) were purchased from BD Biosciences (San Diego, CA). Anti-TIM-3-PE (344823) was purchased from R&D systems. Anti-LAG-3-FITC (17B4) was purchased from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA). Anti-CD107a-PE-Cy7 (H4A3) and anti-perforin-PerCP-Cy5.5 (δG9) were purchased from BD Biosciences. Anti-galectin-9 (ab69630) was purchased from Abcam. Control rabbit IgG (BA-1000) was purchased from Vector Labs (Burlingame, CA). Goat-anti-Rabbit IgG-HRP was purchased from Dako (Carpinteria, CA). All antibodies were used per the manufacturer's recommendations.
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3

Evaluating Memory CD8 T Cell Responses

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C57BL/6 mice received 5 × 105 OT-I CD8+ T cells 1 day before inoculation in the footpad with PBS, 5 μg of rlipo-OVA, 5 μg of rOVA, and 5 μg of rOVA formulated with equal molar Pam3CSK4. To evaluate whether the lipidated antigen elicited specific memory CD8 T cell phenotypes, the lymphocytes were pooled from the popliteal, and inguinal lymph nodes and splenocytes were isolated from the spleens from the immunized mice 28 days after immunization. The Fc receptors were blocked with CD16/CD32 (clone 93, eBioscience), and the cells were stained with anti-CD27-FITC (clone LG·7F9, eBioscience), anti-CD44-FITC (clone IM7, BD Bioscience), anti-Va2TCR-PE (clone B20.1, BD Bioscience), anti-CD43 PE-Cy7 (clone 1B1.1, Biolegend), anti-CD127 PerCP-Cy5.5 (clone A71234, eBioscience), anti-CD62L-APC (clone MEL-14, BD Bioscience) and anti-CD8a-APC/Cy7 (clone 53–6.7, BD Bioscience). The acquisitions were determined using the LSRII flow cytometer (BD Bioscience).
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4

Lung T-cell Phenotyping using Flow Cytometry

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Lung T-cell phenotypes were assessed as described [19 (link)]. 2 x 106 lung cells/well were surface-stained with anti-CD3-eFluor450, anti-CD8-APC-Cy7, anti-CD62L-PE-Cy7, anti-CD69-PE, anti-CD127-PerCP-Cy5.5 (eBiosciences, San Diego, CA), and Live/Dead fixable green viability stain Vivid for 488 nm excitation (Invitrogen). The following tetramer was obtained through the NIH Tetramer Facility: NP147–155-H2-Kd Tetramer-APC (TYQRTRALV) (Atlanta, GA). 50,000 events per sample were acquired on an LSRII flow cytometer.
FACS Diva V6 software (BD Biosciences, San Jose, CA) was used for data acquisition and FlowJo V7.6.5 (TreeStar, Ashland, OR) for data analysis and display. Single color-stained cells were used for compensation, and fluorescence minus one (FMO) controls were used for gate setting.
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