Automated CSF leukocyte counts were performed on ABX micros 60 (Horiba, Montpellier, France). Cells were washed twice with phosphate-buffered saline and immune-stained for 30 min at 4 °C with the following panel of antibodies: anti-CD45-BV786, anti-CD8-BV510, antiCD66b-BV421, anti-CD45-PerCp5.5 (BD Bioscience, San Jose, CA, USA); anti-CD3-FITC, anti-CD19-PECy7, anti-CD11b-PECy5, anti-CD161-PE, anti-CD14-Alexa700, anti-CD11c-APC (Tonbo Biosciences, San Diego, CA, USA). Optimal antibody concentrations were previously defined by titration20 (link). The cells were washed and suspended in 1% paraformaldehyde. Flow cytometry data was collected on a BD FACS LSR flow cytometer equipped with four lasers. Analyses of leukocyte subsets on CSF and PB samples were conducted by 13 flow cytometry within 30 min from collection. BD FACS Diva software was used for data acquisition and Flow Jo v10 (Becton–Dickinson Bioscience, San Jose, CA, USA) was used for data analysis. The gating strategy is depicted in Supplemental Fig. 2 .
Anti cd8 bv510
Manufactured by BD
Sourced in United States
The Anti-CD8-BV510 is a fluorochrome-conjugated monoclonal antibody that binds to the CD8 antigen expressed on the surface of certain T lymphocytes. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (3 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Anti ifn γ apc, by BD (2 mentions)
Anti cd3 percp, by BioLegend (2 mentions)
Anti tnfα pecy7, by BD (2 mentions)
Live dead near ir fluorescent reactive dye, by Thermo Fisher Scientific (2 mentions)
Anti cd27 bv605, by BD (2 mentions)
Anti cd4 bv786, by BD (2 mentions)
Anti cd45 bv786, by BD (1 mentions)
Anti cd11c apc, by Cytek Biosciences (1 mentions)
Facs lsr flow cytometer, by BD (1 mentions)
Abx micros 60, by Horiba (1 mentions)
Anti cd3 apc cy7, by BD (1 mentions)
Anti cd4 bb700, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Anti ki67 fitc, by BD (1 mentions)
Anti cd38 pe, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti hla dr bv421, by BD (1 mentions)
11 protocols using anti cd8 bv510
1
Automated CSF Leukocyte Analysis by Flow Cytometry
2
Immune Cell Phenotyping by Flow Cytometry
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Two milliliters of peripheral blood were collected from participants and placed in an ethylene diamine tetraacetic acid (EDTA) anticoagulant tube for routine blood tests during recruitment. Another 100 μL of peripheral blood was taken to be labeled with antibodies. After antibody labeling, the red blood cells were lysed and washed with phosphate-buffered saline. The antibody labels were then detected. The labeled antibodies were described in previous literature [25 (link)] as follows: anti-CD3-allophycocyanin (APC)-cyanine (Cy) 7 (clone SK7), anti-CD4-BB700 (clone SK3), anti-CD8-BV510 (clone SK1), anti-CD25-phycoerythrin (PE) (clone M-A251), anti-CD127-BV421 (clone HIL-7R-M21), anti-CD62L-APC (clone DREG-56), anti-CD45RO-BB515 (clone UCHL1), and anti-HLA-DR-PE-Cy7 (clone G46-6). One hundred microliters of peripheral blood were also collected from the homotypic controls for antibody labeling. Homotypic controls were tested using anti-CD3-APC-Cy7 (clone SK7), anti-CD4-BB700 (clone SK3), anti-CD8-BV510 (clone SK1), immunoglobulin 1 (IgG1) k-PE (clone MOPC-21), IgG1 k-BV421 (clone X40), IgG1 k-APC (clone MOPC-21), IgG2a k-BB515 (clone G155-178), and IgG2a k-PE-Cy7 (clone G155-178) (BD Biosciences).
Flow cytometry BD FACS Canto™ II was applied for data collection and FlowJo V10 software (Tree Star, Inc., Ashland, OR, USA) was utilized for data analysis.
Flow cytometry BD FACS Canto™ II was applied for data collection and FlowJo V10 software (Tree Star, Inc., Ashland, OR, USA) was utilized for data analysis.
3
Comprehensive Multiparametric Phenotyping of PBMCs
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After stimulation, PBMCs were labeled with viability marker LIVE/DEAD Near-IR fluorescent reactive dye (Thermo Fisher) for 30 min and subsequently stained for 20 min with the following surface markers: anti-CD3-PerCP (BioLegend), anti-CD4-BV786, anti-CD8-BV510, anti-CD27-BV605, anti-CD38-PE, and anti-HLA-DR-BV421 (BD Bioscience). Cells were then fixed and permeabilized with the Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher) and stained for 30 min with intracellular markers: anti-IFNγ-APC, anti-TNFα-PE-Cy7 and anti-Ki-67-FITC (BD Bioscience). The complete list of antibodies, conjugated proteins and dilutions can be found in Supplementary Table 1 . All incubation processes were performed at room temperature in darkness. Fluorescence Minus One (FMO) controls of the four analyzed cell markers (CD27, CD38, HLA-DR, and Ki-67) were included in each run to accurately distinguish negative from positive populations. Samples were resuspended in 100 μl of PBS-0.1%BSA (Bovine Serum Albumin, Sigma Aldrich) and acquired in a BD LSRFortessa flow cytometer (BD Bioscience) using FACSDiva software (BD Biosciences) with compensated parameters.
4
T Cell Proliferation Induced by ALP-treated DCs
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To investigate the T cell proliferation induced by ALP-treated DCs, we separated splenic CD4+ and CD8+ T cells from BALB/c mice using Miltenyi MACS microbeads conjugated with anti-CD4 or anti-CD8 mAbs (Biotec, San Diego, CA, USA) and a MACS large separation column (Miltenyi Biotec). The isolated cells (2 × 106 cells/well) were next stained with carboxyfluorescein succinimidyl ester (CFSE, 1 μM, Invitrogen) and co-cultured with each set of DCs (ALP- and LPS-treated DCs, 2 × 105 cells per well). After 2 days of co-culture, the T cells were stained with anti-CD4 (Alexa488, BD Bioscience) and anti-CD8 (BV510, BD Bioscience) mAbs. The stained cells were counted using FACSverse, and the FACS data were analyzed with the FlowJo V10 software. Supernatants were collected to measure the IFN-γ (BD Bioscience), IL-2 (eBioscience), and IL-5 (eBioscience) levels using ELISA.
5
Modulation of T Cell Activation by Anti-TNF Agents
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PBMCs from healthy donors were isolated by Ficoll-Hypaque gradient separation (Lymphoprep). PBMC were stimulated with 0.5 µg/ml of plate-bound anti-CD3 mAb (OKT-3; eBioscience) and 1 µg/ml of soluble anti-CD28 mAb in RPMI-10% FBS supplemented with IL-2 (20 U/ml, Proleukin) in the presence or absence of 10 μg/ml of anti-hTNF (Infliximab, Janssen), 4 μg/ml of Etanercept (Sandoz Farma), 1 μg/ml anti-TNFR1 (clone: 55R-170, ThermoFisher Scientific) or 1 μg/ml anti-TNFR2 (clone: 2222.311, ThermoFisher Scientific). Seven days later, cells were re-plated into fresh anti-CD3/CD28 mAbs coated plates and cultured overnight in the same conditions. Cells were analyzed by multicolor flow cytometry (BD FACSCanto™ II system; Zombie NIR, anti-CD3 PeCy7 BD, anti-CD8 BV510, anti-CD4-PE, Annexin V-FITC, anti-CD25-BV421, and anti-PD-1 PerCPCy5.5 (BioLegend). All analyses were performed using Flowjo software (Tree Star).
6
Intracellular Cytokine Staining Protocol
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We used intracellular cell staining (ICS) to detect intracellular cytokines as previously reported 29 (link). First, cells were stimulated by addition of phorbol myristate acetate (PMA; 50 ng/mL, eBioscience, San Diego, California, USA) with ionomycin (2 μg/mL, eBioscience) in the presence of brefeldin A (10 μg/mL, eBioscience) for 4 hours at 37°C. Cells were subsequently stained with extracellular anti-CD3-Percp-cy5.5, anti-CD4-APC-H7, anti-CD8-BV510 (all BD Biosciences, San Jose, California, USA) for 20 min, followed by a fixation and permeabilization procedure using Perm/Wash Buffer (BD Biosciences). Thereafter, cells were washed with permeabilization buffer and incubated with intracellular antibodies including anti-IFNγ-Alexa 488, anti- IL4-BV711, anti-IL-9-PE, and anti-IL17-BV650 (all BD Biosciences). Appropriate isotypes were used. Finally, stained cells were acquired on an LSRII Fortessa cytometer (BD Biosciences). Data were analyzed using Flowjo software (Ashland, OR, USA).
7
Characterizing T Cell Phenotypes by Flow Cytometry
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After stimulation, PBMCs were stained with the following surface antibodies: anti-CD4 BV786 (BD Bioscience; clone SK3), anti-CD3 PerCP (BioLegend, San Diego, CA, USA; clone SK7), anti-CD27 BV605 (BD Bioscience; clone L128), anti-CCR4 PE-CF594 (BD Bioscience; clone 1G1), and anti-CD8 BV510 (BD Bioscience; clone SK1). A viability marker was also used to exclude dead cells (LIVE/DEAD, Near-IR fluorescent reactive dye; Thermo Fisher, Waltham, MA, USA). For intracellular staining, PBMCs were fixed/permeabilized (IntraStain; Dako, Santa Clara, CA, USA) and then stained with anti-IFN-γ APC (BD Bioscience; clone B27) and anti-TNF-α PE-Cy7 (BD Bioscience; clone Mab11). Markers detection was performed in a BD LSRFortessa flow cytometer (BD Bioscience). A total of 100.000 alive CD3+ T-cells were acquired within 2–3 h after staining.
8
Activin A Modulates PBMC Activation Markers
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Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples anti-coagulated with EDTA using Ficoll-Hypaque density gradient centrifugation. Cells were seeded in 12-well plates at a density of 1 × 106 cells per well. The cells were then grown in complete RPMI 1640 medium supplemented with 2% FCS and stimulated with or without 5 ng/ml activin A for 24 h in a 5% CO2 incubator. To detect the expression of ActRIIA, cells were stained with anti-CD4-eFloure450 (BD Biosciences), anti-CD8-PE-Cy7 (BD Biosciences), anti-CD19-PE (BD Biosciences) and anti-ActRIIA-APC (BD Biosciences) antibodies for 30 min at RT. To detect the expression of CD25 and CD69, cells were stained with anti-CD4-PE-Cy7 (BD Biosciences), anti-CD8-BV510 (BD Biosciences), anti-CD19-BV421 (BD Biosciences), anti-CD25-PE (BD Biosciences) and anti-69-FITC (BD Biosciences) antibodies for 30 min at RT. The labelled cells were analysed by flow cytometry (BD FACS Canto II). The data were collected and analysed with FlowJo v10 to assess the percentages of fluorescence-positive cells.
9
Immunophenotyping of T cell subsets
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Immunophenotyping of peripheral blood CD3+T cell subsets (CD4+ and CD8+, CD4+CD45RA+, CD4+CD45RA−, CD8+CD45RA+, CD8+CD45RA−, NKT), NK cells (CD3−CD16+CD56+) cells and B lymphocytes (CD19+) were by a multiparametric flow cytometry analysis at each time point: pre-infusion and weekly during two weeks after infusion.
Peripheral blood was stained for surface markers with the following fluorochrome-conjugated anti-human antibodies (BD Bioscience): anti-CD45 APC, anti-CD3 FITC, anti-CD4 PerCP, anti-CD8 Bv510, anti-CD19 PeCy7, anti-CD45RA APC—H7, anti-CD16 PE, and anti-CD56 PE. Cells were then acquired by flow cytometry on FACSCanto™ II (BD Biosciences). The analysis was performed with BD FACSDiva™ Software v8.0.2 (BD Bioscience).
Peripheral blood was stained for surface markers with the following fluorochrome-conjugated anti-human antibodies (BD Bioscience): anti-CD45 APC, anti-CD3 FITC, anti-CD4 PerCP, anti-CD8 Bv510, anti-CD19 PeCy7, anti-CD45RA APC—H7, anti-CD16 PE, and anti-CD56 PE. Cells were then acquired by flow cytometry on FACSCanto™ II (BD Biosciences). The analysis was performed with BD FACSDiva™ Software v8.0.2 (BD Bioscience).
10
T Cell Immunophenotyping and Viability
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PBMCs or T cells were stained with PE-Cy7 anti-CD3 (BD, 560910), APC-Cy7 anti-CD4 (BD, 557871), BV510 anti-CD8 (BD, 344732) for T cell subset analysis. Propidium iodide (PI, 556547, BD Bioscience) were used for cell death analysis according to manufacturer’s instruction. Data were acquired using the flow cytometer (BD verse) and analyzed with the Flowjo software.
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