Fresh samples were analyzed at diagnosis and/or at different time points during the follow-up. Ten-color labeling was performed with the following antibodies: anti–CD3-APCR700, anti–CD26-FITC, anti–CD30-APC, anti–CD279-BV421 (BDBiosciences), anti–CD158ek-PE (Miltenyi Biotec), anti–CD4-PC7, anti–CD7-PerCP-Cy5.5, anti–CD28-PE-CF594, anti–CD45-BV510 (BioLegend), and anti–CD8-AA750 (Beckman Coulter). Anti–CD158ek-PE was chosen because of its commercial availability. There is no evidence of KIR3DL1 (CD158e) expression on either Sezary cells or normal T CD4+.14 (link) Anti–T-cell receptor (TCR)-Vβ antibodies (Beckman Coulter) were used when clonal circulating cells were suspected. Red blood cells were lysed with BD FACS Lysing Solution (BDBiosciences). Acquisition of a minimum of 50 000 T cells was performed using Navios Flow Cytometer (Beckman Coulter). Counting beads were used for absolute values assessment (FlowCount Fluorospheres; Beckman Coulter). Data were analyzed using Kaluza software (Beckman Coulter).
Anti cd3 apcr700
Manufactured by BD
Anti–CD3-APCR700 is a lab equipment product. It is a fluorescently-labeled antibody that binds to the CD3 cell surface receptor, which is expressed on T cells. The antibody is conjugated to the APC-R700 fluorophore, allowing detection and analysis of CD3-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd19 apc, by BD (2 mentions)
Anti cd8 bv786, by BD (2 mentions)
Anti cd4 bv711, by BD (2 mentions)
Navios flow cytometer, by Beckman Coulter (1 mentions)
Anti cd45 bv510, by BioLegend (1 mentions)
Facs lysing solution, by BD (1 mentions)
Flow count fluorosphere, by Beckman Coulter (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Anti cd56 bv510, by BD (1 mentions)
Anti cd107a bv421, by BioLegend (1 mentions)
Anti cd45ro pe cy7, by BD (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Facsmelody sorter, by BD (1 mentions)
Ccr7 percp cy5, by BioLegend (1 mentions)
L4144, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Human ab serum, by Merck Group (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
4 protocols using anti cd3 apcr700
1
Multicolor Flow Cytometry Analysis of T-cell Subsets
2
Characterizing Activated T-Cell Subsets
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Cells were labeled with fluorochrome-conjugated antibodies, anti-CD3 APC-R700, anti-CD4 BV711, anti-CD8 BV786, anti-CD56 BV510, and anti-CD19 APC and with fixable viability stain eF780 (all BD). Anti-CD107a BV421 (BioLegend) was added during culture. For intracellular staining, cells were fixed, permeabilized, and stained with phycoerythrin-conjugated anti-CD137 and allophycocyanin-conjugated anti-IFN-γ (BD) using the Foxp3/transcription factor staining buffer set (eBioscience) according to the manufacturer’s protocol. Cells were acquired on an LSRFortessa X20 flow cytometer (BD). Data were analyzed using FlowJo (v10; Tree Star). The results are expressed as the percentage of CD137+ or CD107a+ cells in the CD3+ CD4+ live gate.
3
Generating Effector CD8+ T Cell Lines
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In order to generate effector CD8+ T cell lines, PBMCs from three HLA-B*07:02 positive and three HLA-A*01:01 positive mumps patients were thawed. Cells were stained with fluorochrome-conjugated antibodies anti-CD3-APC/R700 (659,119, BD), anti-CD4-BV711 (563,028, BD), anti-CD8-BV786 (563,823, BD), anti-CD45RO-PE/Cy7 (560,608, BD), and CCR7-PerCP/Cy5.5 (B286436, BioLegend). A subset of the effector T cells (CD3+, CD8+, CCR7−, CD45RO−) was sorted using FACSMelody sorter (BD) in order to increase MuV-specific T cell frequencies for peptide immunogenicity testing. Sorted T cells were further expanded in AIM-V medium (12,055–083, Gibco) supplemented with 2% human AB serum (H6914, Sigma), with irradiated (3000 rad) allogeneic PBMCs and irradiated (5000 rad) allogeneic BLCL cells, 1 μg/ml phytohemagglutinin (L4144, Sigma), and 5 ng/ml IL-2 (130–097-743, Miltenyi Biotec). Every 3–4 days IL-2 (5 ng/ml) was added. Cells were frozen when they reached a total of ≥ 2 × 106.
4
Comprehensive Immune Cell Profiling
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To evaluate the relative proportions of lymphocyte populations in peripheral blood, the following panel of fluorochrome conjugated antibodies was used: anti CD45 BUV 395, anti CD3 APC R700, anti CD19 APC, anti CD16 PE-Cy7, anti CD56 BB515, anti CD4 BUV 7373, anti CD8 BV 785, anti CD25 BV 421, anti CD127 BB700, anti CD45RA BV605, anti CD197 BV711, anti FoxP3 PE, anti RORγT BV650 (all from BD Biosciences, Milan, Italy). Blood samples were stained with anti membrane associated molecules and, after incubation, erythrocytes were lysed using FACS lysing solution, according to manufacturer’s instruction. To analyze the expression of transcription factors (FoxP3 and RORγT), samples were fixed and permeabilized using Transcription Facytors Buffer Set (BD Bioscience), according to standard protocol. Samples were acquired and analyzed on a LSR Fortessa X-20 (Becton Dickinson), using FACS DIVA v8.0.2 software.
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