Lung cells were stained in PBS/1% FCS/0.01% sodium azide. Cells were incubated with rabbit serum (Sigma-Aldrich) for 15 min before staining with FITC-T1ST2 (Morwell Diagnostics, Zurich, Switzerland), PE-CD8 (BD Biosciences, Oxford, UK), PerCP-Cy5.5-CD4 (eBioscience, Hatfield, UK) or APC-CD3e (BD Biosciences) or relevant isotype controls for 20 min at 4°C. Cells were washed twice and fixed in fixation buffer (eBioscience). For intracellular cytokine staining, cells were stimulated with PMA (Sigma-Aldrich)/Ionomycin (Emdchemicals Inc, San Diego, CA, USA) in the presence of Brefeldin A (Sigma-Aldrich) for 3 h before extracellular staining. Thereafter, cells were permeabilized using permeabilization buffer (eBioscience) and stained with PE-IL-10 (BD Pharmingen), or appropriate isotype control. Analysis was performed using a FACS ARIA (BD Biosciences) and FlowJo software (v7.6.5; Tree Star, Ashland, OR, USA).
Apc cd3e
Manufactured by BD
Sourced in United States
The APC-CD3e is a fluorochrome-conjugated antibody used for the identification and enumeration of T lymphocytes by flow cytometry. It binds to the CD3 epsilon chain, a component of the T cell receptor complex, allowing the detection of T cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b pe, by BD (3 mentions)
Cd3 fitc, by BD (2 mentions)
Fc block anti cd16 32, by BD (2 mentions)
Cd19 pe, by BD (2 mentions)
Cd19 fitc, by BD (2 mentions)
Percp cy5.5 cd45, by BD (2 mentions)
Percp cy5.5 mhcii, by BD (2 mentions)
Percp cy5.5 cd4, by BD (2 mentions)
Cd25 apc, by BD (2 mentions)
Cd4 pe, by BD (2 mentions)
Cd19 pe cy7, by BD (2 mentions)
Cd11b pe cy7, by BD (2 mentions)
Cd45 apc cy7, by BD (2 mentions)
Foxp3 pe, by BD (2 mentions)
Cd40 fitc, by BD (2 mentions)
Percp cd3e, by BD (2 mentions)
Percp cy5.5 cd25, by BD (2 mentions)
Cd138 apc, by BD (2 mentions)
Fitc il10, by BD (2 mentions)
Fitc mhc 2, by BD (2 mentions)
6 protocols using apc cd3e
1
Flow Cytometry Analysis of Lung Cells
2
Characterization of T-cell Populations
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The cells were stained for surface and intracellular/intranuclear, and analysis by FACS Canto flow cytometer (BD Biosciences). The following fluorochrome conjugated Abs: PerCP Cy5.5-CD45, APC Cy7-CD45, PerCP Cy5.5-CD4, PE-CD4, FITC-CD4, FITC-CD3, APC-CD3e, PerCP-CD3e, APC Cy7-CD3, PE-CD11b,PE Cy7-CD11b, FITC-MHC-II, PerCP Cy5.5-MHCII, PE-CD19, FITC-CD19, PE Cy7-CD19, APC-CD25, PE Cy7-CD25, PerCp Cy5.5-CD25, APC-CD138, FITC-CD40, PE-FoxP3, FITC-IL10 and anti-CD16/32 (Fc block) were purchased from BD Pharmingen (San Diego, CA), Biolegend (San Diego, CA), or eBiosciences (Thermo Fisher Scientific, MA, USA). Frequencies and numbers of populations in the meninges, lymph nodes and spleens of B6 and BTBR mice were gated based on FSC-A and SSC-A, followed by gating in single cells and finally gated on CD45+ cells. Acquired FCS files were analyzed by Flow Jo-V10. Treg cell population were identified based on CD25+FoxP3+ cell in CD3+CD4+ population. To observe the Treg cell population, CD4+ cells were analyzed for the expression of CD25 and FoxP3 and gated on the double positive population. Data were obtained from three to four independent experiments with 4–6 pairs of mice in each experiment. In each experiment, equal number (2/3 males and 2/3 females from each group) of male and female mice were used.
3
Comprehensive Immune Cell Profiling
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The cells were stained for surface and intracellular/intranuclear, and analysis by FACS Canto flow cytometer (BD Biosciences). The following fluorochrome conjugated Abs: PerCP Cy5.5-CD45, APC Cy7-CD45, PerCP Cy5.5-CD4, PE-CD4, FITC-CD4, FITC-CD3, APC-CD3e, PerCP-CD3e, APC Cy7-CD3, PE-CD11b,PE Cy7-CD11b, FITC-MHC-II, PerCP Cy5.5-MHCII, PE-CD19, FITC-CD19, PE Cy7-CD19, APC-CD25, PE Cy7-CD25, PerCp Cy5.5-CD25, APC-CD138, FITC-CD40, PE-FoxP3, FITC-IL10 and anti-CD16/32 (Fc block) were purchased from BD Pharmingen (San Diego, CA), Biolegend (San Diego, CA), or eBiosciences (Thermo Fisher Scientific, MA, USA).
Frequencies and numbers of populations in the meninges, lymph nodes and spleens of B6 and BTBR mice were gated based on FSC-A and SSC-A, followed by gating in single cells and finally gated on CD45 + cells. Acquired FCS files were analyzed by Flow Jo-V10.
Frequencies and numbers of populations in the meninges, lymph nodes and spleens of B6 and BTBR mice were gated based on FSC-A and SSC-A, followed by gating in single cells and finally gated on CD45 + cells. Acquired FCS files were analyzed by Flow Jo-V10.
4
Multicolor Flow Cytometry of Leukocytes
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To characterise circulating leukocytes by flow cytometry, 10 µL of heparinised whole blood was incubated for 30 min at RT with CD45-FITC (BD Biosciences, Franklin Lakes, NJ, USA) to identify leukocytes. Lymphocytes and neutrophils were identified by complexity and morphology, whereas monocytes were gated based on the CD115-APC marker (Biolegend, San Diego, CA, USA). Ly6C-PerCP (BD Biosciences, San Diego, CA, USA) and CD115-APC (Biolegend, San Diego, CA, USA) were used for Ly6Clow and Ly6Chi-monocyte subsets identification. For circulating lymphocytes analysis, 10 µL of heparinised whole blood was incubated with 5 µL Brilliant Stain Buffer (BD Biosciences, San Diego, CA, USA) followed by CD4-BV421, CD8a-BV510, CD3e-APC, or CD69-PE antibodies (BD Biosciences, San Diego, CA, USA). Mouse Tregs were identified using Kit FoxP3 Staining Buffer Set and with anti-CD25-APC, anti-Foxp3-PE, and anti-CD4-VioBlue (all from Miltenyi, Bergisch Gladbach, Germany). The samples were incubated with FACS Lysing solution (BD Biosciences) for 10 min before flow cytometry analysis (FACSVerse or FACS Fortessa Flow cytometer, BD Biosciences, San Diego, CA, USA).
5
Multicolor Flow Cytometric Analysis
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Blood was washed in PBS, stained with live/dead fixable blue dead cell staining kit (Invitrogen), washed in PBS and blocked in 2% FBS-PBS / 0.02% sodium azide plus Fc-block (Anti-CD16/32 antibody 1:200, BD Biosciences). Surface antigens were detected by incubation for 30 min at 4°C with conjugated antibodies including CD45.1-eFluor 450 (eBioscience Cat# 48-0453-82, RRID:AB_1272189 ), CD45.2-FITC (BD Biosciences Cat# 553772, RRID:AB_395041 ), CD3e-APC (BD Biosciences Cat# 561826, RRID:AB_10896663 ), CD19-APC-H7 (BD Biosciences Cat# 560143, RRID:AB_1645234 ), CD11b-PE (BD Biosciences Cat# 562287, RRID:AB_11154216 ) and GR1-Alexa Fluor 700 (BioLegend Cat# 108422, RRID:AB_2137487 ). Following washing with 2% FBS-PBS 0.02% sodium azide, red cells were lysed and leukocytes fixed by incubating in lyse/fix solution (BD Biosciences). Cells were washed with PBS and analyzed on an LSR-II cytometer (Becton Dickenson). Data were processed using FlowJo Software (Tree Star).
6
Multiparametric Flow Cytometry Analysis
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Peripheral blood counts were acquired using the V-Sight hematology analyzer (A. Menarini Diagnostic). RBCs were lysed for 5 min at RT in RBC lysis buffer. Freshly isolated BM, spleen, thymus, lymph node, and white blood cells were stained with Fixable Viability Dye eFluor 780 (eBioscience), blocked for 10 minutes with TruStain FcX anti-mouse CD16/32 (BioLegends) and stained for 30 minutes in brilliant stain buffer (BD Bioscience) with antibodies against Ly5.1-PerCP-Cy5.5 (eBioscence #45-0453), Ly5.2-V500 (BD Bioscence 562129), CD19-BV786 (BD Bioscience #563333), CD3e-APC (BD Bioscence #553066), Mac1-BV650 (BD Bioscience #563402), Gr-1-APC (eBioscience #17-5931) and Ter119-BV421 (BD Bioscence 563998). Cells were analyzed by FACSFortessa (BD BioScience) and FlowJo software.
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