One milliliter of blood collected in sodium heparin tubes was stimulated with 10 ng IFNβ or unstimulated for 24 h. Proteomic Stabilizer PROT1 (Smart Tube, Inc.) was added and incubated for 10 min at room temperature prior to freezing at −80°C. Samples were thawed, and single‐cell suspensions of blood leukocytes were obtained using Thaw‐Lyse buffer (Smart Tube, Inc) according to manufacturer's instructions. Following incubation with human Fc receptor blocker, TruStain FcX (Biolegend Inc.), and Super Bright Complete Staining Buffer (Thermo Fisher), cells were stained with fluorescently conjugated antibodies: CD14 BV421, CD64 BV785, HLA‐DR FITC, Siglec‐1 PE, CD38 APC, CD19 BV650, CD66b PerCP/Cy5.5, CD16 APC/Cy7, CD86 PE/Cy7, and CD3 BV510 from Biolegend, acquired with a Cytek Northern Lights 3000 (Cytek Biosciences) and analyzed using FlowJo.
Cd14 bv421
Manufactured by BioLegend
Sourced in United States
CD14-BV421 is a fluorochrome-conjugated antibody that detects the CD14 cell surface protein. CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed primarily on monocytes and macrophages and functions as a co-receptor for the detection of bacterial lipopolysaccharide (LPS).
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Lab products found in correlation
Cd3 bv510, by BioLegend (2 mentions)
Trustain fcx, by BioLegend (2 mentions)
Cd16 apc cy7, by BioLegend (2 mentions)
Cd86 pe cy7, by BioLegend (2 mentions)
Northern lights 3000, by Cytek (2 mentions)
Super bright complete staining buffer, by Thermo Fisher Scientific (2 mentions)
Hla dr fitc, by BioLegend (2 mentions)
Cd64 bv785, by BioLegend (2 mentions)
Siglec 1 pe, by BioLegend (2 mentions)
Cd66b percp cy5, by BioLegend (2 mentions)
Cd19 bv650, by BioLegend (2 mentions)
Cd38 apc, by BioLegend (2 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions)
Methylcellulose medium, by STEMCELL (2 mentions)
Alexa fluor 488 anti human cd41 antibody, by BioLegend (2 mentions)
Apc anti human cd235ab antibody, by BioLegend (2 mentions)
Cd66b alexa594, by BioLegend (2 mentions)
Efluor 506, by Thermo Fisher Scientific (2 mentions)
Cd19 v500, by BD (2 mentions)
Lsrii cytometer, by BD (2 mentions)
10 protocols using cd14 bv421
1
Multiparametric Analysis of IFNβ-Stimulated Leukocytes
2
Single-cell hematopoietic colony assay
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Antibody list: Alexa Fluor® 488 anti-human CD41 Antibody (Biolegend, cat# 303723), APC anti-human CD235ab Antibody (Biolegend, cat# 306607), CD66b-Alexa594 (Biolegend, cat# 392907), CD14-BV421 (Biolegend, cat# 325627).
Single hemogenic endothelial cell was seeded in fibronectin coated 96-well plates (Cellvis, cat# P96–1.5P) as described above. Cells were cultured in 100 μL of methylcellulose medium (STEMCELL Technologies, cat# 4435) per well for 8 days before staining for CFU-E and BFU-E, for 14 days before staining for CFU-GM and CFU-GEMM. For staining, antibodies were 1:100 diluted in IMDM (GIBCO, cat# 12440046). 30 μL of diluted antibodies was added to each well on top of the methylcellulose medium (pipette slowly so as not to disturb the hematopoietic colonies). Incubate plates at 37°C overnight before imaging with fluorescence microscope.
Single hemogenic endothelial cell was seeded in fibronectin coated 96-well plates (Cellvis, cat# P96–1.5P) as described above. Cells were cultured in 100 μL of methylcellulose medium (STEMCELL Technologies, cat# 4435) per well for 8 days before staining for CFU-E and BFU-E, for 14 days before staining for CFU-GM and CFU-GEMM. For staining, antibodies were 1:100 diluted in IMDM (GIBCO, cat# 12440046). 30 μL of diluted antibodies was added to each well on top of the methylcellulose medium (pipette slowly so as not to disturb the hematopoietic colonies). Incubate plates at 37°C overnight before imaging with fluorescence microscope.
3
Isolation and Expansion of Donor NK Cells
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Fresh healthy donor NK cells were purchased from AllCells with either CD56 positive selection or CD56 negative selection (Allcells, cat#PB012-P or PB012-N). For 2D migration experiments, NK cells were enriched from peripheral blood using RosetteSep (StemCell Technologies) from healthy adult donors. T cells, B cells and monocytes were isolated from PBMCs (AllCells) using Mojosort magnetic cell separation system from Biolegend via CD3 positivity (Biolegend, cat#480133), CD19 positivity (Biolegend, cat#480105), CD14 positivity (Biolegend, cat#480093). PBMC purity was assessed using flow cytometry: CD3-APC (Biolegend, cat#300411), CD14-BV421 (Biolegend, cat#325627), CD45-FITC (BD Bioscience, cat#347463), CD56-PE (BD Bioscience, cat#555516), CD20-PE (BD Bioscience, cat#555623). For donor NK cell lysis of PANC-1 clusters, primary donor NK cells were purchased from AllCells then expanded using irradiated K562–4-1BBL-mbIL-21 (names “CSTX002”) cells kindly provided by Dr. Dean Lee according to his protocol58 (link).
4
Isolation and Characterization of CD1a+ Cells
5
Multiparametric Phenotyping of MAIT Cells
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PBMCs were stained with 1:100 MR1 5-OP-RU or 6-FP (as a control) tetramer for 40 min at room temperature. After 40 min, incubation cells were also stained with fixable viability dye eFluor 506 (eBioscience) and with combinations of anti-human CD3-Alexa Fluor 700 (UCHT1; BD Pharmingen), CD4-allophycocyanin-eFluor 780 (RPA-T4; eBioscience), CD8-BV650 (RPA-T8; BioLegend), Vα7.2-PE-Cy7 (3C10; BioLegend), CD161-allophycocyanin (HP-3G10; eBioscience), TIGIT-PerCP-eFluor710 (MBSA43; eBioscience), CXCR6-PE Dazzle594 (K041E5; BioLegend), CCR1-PerCP Cy5.5 (5F10B29; BioLegend), CD243-BV421 (UIC2; BD Biosciences), CXCR4-PE Dazzle 594 (12G5; BioLegend), CD127-FITC (eBioRDR5; eBioscience), CD14-V500 (M5E2; BD Biosciences), CD14-BV421 (HCD14; BioLegend), and CD19-V500 (HIB19; BD Biosciences) for 30 min at room temperature. After two washes in PBS, cells were resuspended in PBS and cells were acquired on a LSRII Cytometer (Becton Dickinson).
6
Single-cell hematopoietic colony assay
7
LPS-induced CD42b expression on neutrophils
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CD42b receptor transfer to neutrophils and monocytes cell surface was investigated by flow cytometry using a Beckman Coulter Cytoflex (Beckman). Citrated blood from five healthy donors was incubated with 0.5 μg/ml E. coli O111:B4 LPS (Sigma-Aldrich®) for 15 min at 37 °C. 100 μl of blood was used and red blood cells were lysed with a BD Phosflow™ Lyse/Fix 5X. After lysis, samples were washed once with 0.5% BSA in PBS and incubated with CD66b-FITC (Clone: G10F5, BD Bioscience), CD42b-PE-Cy™5 (Clone: HIP1, BD Bioscience) and CD14-BV421 (Clone: HCD14, BioLegend®) antibodies were added (1:50) to the cell suspension and incubated for 60 min at 37 °C in the dark. CD42b is platelet-specific surface protein as claimed by the manufacturer. To exclude that other cells can up-regulate CD42b upon stimulation, isolated neutrophils were stimulated with LPS. For one donor, the manual gating analysis using FMO, and gating controls was carried out. Samples were washed once with 0.5% BSA in PBS and the cell pellet was resuspended in 300 μl of washing buffer. The percentage of CD42b on the cell surface was calibrated with control cells that were not treated with LPS. The results are presented as mean values ± s.e.m.
8
Circadian Protein Profiling in Whole Blood Leukocytes
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For whole blood samples, leukocyte populations were stained with CD3-APC-Cy7 (Biolegend, California, USA, 317342), CD14-BV421 (Biolegend, 301830), and CD16-PerCP-Cy5.5 (Biolegend, 301828) . Cells were fixed with FIX (Nordic-MUbio, Netherlands, GAS-002) and stored in staining buffer (PBS + 2%FBS) overnight at 4°C. On the next day, all samples were contemporaneously permeabilized using PERM (Nordic-MUbio, Netherlands, GAS-002). Samples were then blocked with human TruStain FcX ™ Fc Receptor Blocking Solution (Biolegend, 422302), and circadian proteins were stained intracellularly using the following primary antibodies: NR1D1 (Abcam, United Kingdom, ab174309), REV ERB beta (Novusbio, Colorado, USA, NBP2-19576), BMAL1 (Novusbio, NB100-2288), CLOCK (Mybiosource, California, USA, MBS4750976), ROR alpha (Thermo Fisher Scientific, Massachusetts, USA, PA1-812), ROR beta (Novusbio, NBP1-82532), and ROR gamma-PE (R&D Systems, Minnesota, USA, IC6006P). The secondary donkey anti rabbit-PE antibody (Biolegend, 406421) was used for detection. Samples were measured on a BD FACSCanto II flow cytometer and analyzed as FI-FMO using FlowJo 10.8.1. Afterwards, Z-scores were calculated and normalized to TP 0 of the healthy control.
9
Multicolor Flow Cytometry of Interferon-Stimulated Leukocytes
10
Multiparametric Phenotyping of MAIT Cells
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