Cells were prepared for flow cytometry as previously described [3 (link)] and the following antibodies were used: CD45-APC, CD15-BV605, CD33-PECy7 and Glycophorin A-PE (BD Biosciences, San Jose, CA, USA), CD33-BV421, CD19-PerCPCy5.5, CD14-BV605, CD117-PerCPCy5.5, CD3-PECy7 and FceRI-PE (BioLegend, San Diego, CA, USA).
Cd14 bv605
Manufactured by BioLegend
Sourced in United States
CD14-BV605 is a fluorochrome-conjugated antibody that binds to the CD14 cell surface antigen. CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein that serves as a co-receptor for the detection of bacterial lipopolysaccharide (LPS). The BV605 fluorochrome provides emission in the violet spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 apc, by BD (2 mentions)
Cd3 pe cy7, by BioLegend (2 mentions)
Cd15 bv605, by BD (2 mentions)
Fceri pe, by BioLegend (2 mentions)
Cd19 percp cy5, by BioLegend (2 mentions)
Glycophorin a pe, by BD (2 mentions)
Lsrfortessa sorp, by BD (2 mentions)
Cd33 bv421, by BioLegend (1 mentions)
Cd117 percpcy5, by BioLegend (1 mentions)
Cd33 pe cy7, by BD (1 mentions)
Cd3 percp cy5, by BD (1 mentions)
Cd11b pe, by Thermo Fisher Scientific (1 mentions)
Zombie uv fixable viability dye, by BioLegend (1 mentions)
Cd11c pe cy7, by BioLegend (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Cd45.2 pe, by Thermo Fisher Scientific (1 mentions)
Ly6g bv510, by BD (1 mentions)
Cd56 buv737, by BD (1 mentions)
Hla dr buv395, by BD (1 mentions)
Cd19 bv786, by BioLegend (1 mentions)
5 protocols using cd14 bv605
1
Multicolor Flow Cytometry Panel
2
Multicolor Flow Cytometry of THP-1 and Blood
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Flow cytometry analysis of THP-1 cells and total blood was performed using the following antibodies. Conjugated anti-human SR-B1 APC (Miltenyi Biotec 130-111-237), CD235a APC Cy7 (BioLegend 3,49,115), CD45 BUV805 (BD Biosciences 6,12,892), CD56 BUV 737 (BD Biosciences 6,12,767), CD11c PeCy7 (BioLegend 3,37,216), CD11b PE (eBioscience 12-0118-41), CD3 PerCP Cy5.5 (BD Biosciences 5,60,835), CD19 BV786 (BioLegend 3,02,239), HLA-DR BUV 395 (BD Biosciences 7,40,302), CD14 BV605 (BioLegend 3,01,834). Labeling of mouse blood was done using the following conjugated antibodies: CD45.2 PE (eBioscience 12-0454–82), CD115 PerCPeF710 (eBioscience 46-1,152-82), CD11b BV650 (eBioscience 48-0112–20), Ly6G BV510 (BD Biosciences 5,63,402), CD3 APC-eF780 (BioLegend 47-0032–82). Fc receptors were blocked using FCR blocking reagent (Myltenyi Biotec). Surface membrane staining was performed in PBS FBS 2%. The Zombie UV fixable viability dye (BioLegend 4,23,107) was used to exclude dead cells. Samples were acquired on a Cytoflex (Beckman Coulter) and analyzed with FlowJo 10 (BD Biosciences).
3
Multiparameter Flow Cytometry Immunophenotyping
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Input and migrated cells were analyzed by flow cytometry. After collection, cells were fixed for 10 minutes in 1% paraformaldehyde at room temperature, washed in PBS/0.1 mM EDTA, followed by blocking in mouse IgG. Cells were labeled with anti-human CD45 efluor450, CD8a APC-efluor780 (eBiosciences, San Diego, CA), CD3 Alexa Fluor 647, CD14 BV605 (BioLegend, San Diego, CA), CD19 BV711, CD4 PE-CF594 (BD Biosciences), and CD16 PE (R&D Systems). Data were acquired on a BD LSRFortessa SORP flow cytometer running Diva6, and analyzed in FlowJo 9.7.5 (Tree Star, Ashland, OR).
4
Multiparametric flow cytometry analysis
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Bones and spleens were crushed, cells resuspended and filtered to obtain a single-cell suspension that was analyzed by flow cytometry on a FACSCanto (BD Biosciences). Cells were stained with the following antibodies: CD45-APC, CD10-BV605, CD15-BV605 and glycophorin A-PE (from BD Biosciences) and CD33-BV421, CD19-PerCPCy5.5, CD34-APCCy7, CD68-PE, CD14-BV605, CD117-PECy7, IgM-PE, CD3-PECy7, FceRI-PE and CD25-BV421 (from BioLegend, San Diego, CA, USA). Control cells were stained with matching isotype controls. Sorting of cells was performed on a FACSAria (BD Biosciences).
For intracellular staining of phosphorylated STAT5 (signal transducer and activator of transcription 5), sorted pre-B cells were fixed in 1.6% paraformaldehyde for 10 min at room temperature. Cells were stored in 90% ethanol in −80 °C until analysis. Cells were washed two times in ice cold phosphate-buffered saline before resuspension in phosphate-buffered saline with 2% fetal calf serum. Cells were kept on ice and stained with antibodies against phosphorylated STAT5 (STAT5P-Alexa Flour 647 from BD) or matching isotype controls. Levels of phosphorylated STAT5 are presented as median fluorescence intensity, normalized to the median fluorescence intensity of isotype-stained cells.
For intracellular staining of phosphorylated STAT5 (signal transducer and activator of transcription 5), sorted pre-B cells were fixed in 1.6% paraformaldehyde for 10 min at room temperature. Cells were stored in 90% ethanol in −80 °C until analysis. Cells were washed two times in ice cold phosphate-buffered saline before resuspension in phosphate-buffered saline with 2% fetal calf serum. Cells were kept on ice and stained with antibodies against phosphorylated STAT5 (STAT5P-Alexa Flour 647 from BD) or matching isotype controls. Levels of phosphorylated STAT5 are presented as median fluorescence intensity, normalized to the median fluorescence intensity of isotype-stained cells.
5
Hematopoietic Subset Analysis by Flow
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The flow cytometry panel used to define hematopoietic subsets was composed of CD34-PECy7, CD90-PECY5, CD38-AF488, CD371-PE, CD45Ra-BV421, CD14-BV605, CD42b-AF647, CD10-PEDazzle594, CD235a-PerCPCy5.5 (BioLegend, California), CD15-BUV395, CD41a-APCH7, CD71-BV786 (BD Biosciences, California), and DAPI for dead cell exclusion (ThermoFisher, California). In short, Day 6 cells were harvested, FcR-blocked (FcR binding inhibitor, ThermoFisher), surface-stained, then analyzed in a buffer containing TruCount Control Beads using a BD LSR Fortessa SORP fitted with a high-throughput sampler (BD Biosciences, California). Data was analyzed in Diva 6.0.2 and exported to CSV where after all cell data was transformed to cell per mL then normalized on a per-donor basis to untreated control wells to determine drug impact.
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