Fitc conjugated cd14

Manufactured by BioLegend

Sourced in United States

FITC-conjugated CD14 is a fluorescently labeled antibody that recognizes the CD14 antigen. CD14 is a cell surface receptor that binds to lipopolysaccharide and plays a role in the innate immune response. The FITC (fluorescein isothiocyanate) label allows for the detection and analysis of CD14-expressing cells using flow cytometry or other fluorescence-based techniques.

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Lab products found in correlation

Apc conjugated cd34, by BD (2 mentions) Pe conjugated cd68, by BioLegend (2 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Pe conjugated cd86, by BioLegend (1 mentions) Flow jo ver 10, by BD (1 mentions) Facsverse flow cytometer, by BD (1 mentions) Percpcy5.5 conjugated f4 80, by BioLegend (1 mentions) Pe conjugated cd11b, by BD (1 mentions) Pe cy7 conjugated cd3, by Thermo Fisher Scientific (1 mentions) Fcr blocker, by BD (1 mentions) Facsverse system, by BD (1 mentions) Fitc conjugated mouse igg2a, by Miltenyi Biotec (1 mentions) Clone y1 82a, by BioLegend (1 mentions) Clone rm3 1, by BioLegend (1 mentions) Apc conjugated mouse igg1, by Miltenyi Biotec (1 mentions) Clone 12g5, by BioLegend (1 mentions) Pe conjugated cd73, by BioLegend (1 mentions) Clone hcd14, by BioLegend (1 mentions) Fitc conjugated cd31, by BD (1 mentions)

6 protocols using fitc conjugated cd14

Flow Cytometric Analysis of ARPE-19 Cell Surface Markers

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The cultured ARPE-19 cells were analyzed using flow cytometry. The cells were harvested by treatment with 0.25% trypsin- 0.5% EDTA (Gibco, Carlsbad, CA, USA), washed with 1X PBS, and the cell pellet was resuspended in 2% fetal bovine serum in 1X PBS for 1 h at room temperature.
The identification of surface molecules was carried out by incubating the cells with human monoclonal APC-conjugated anti-toll like receptor 4 (TLR4), FITC-conjugated CD14, and PE-conjugated CD86 antibodies (all from Biolegend, San Diego, CA, USA) for 20 min at 4 °C, according to the manufacturer’s recommendations. Cells were washed with 1X PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, Inc.) for 20 min at room temperature, washed, and acquired with a BD FACSVerse Flow Cytometer (BD Biosciences, San Jose, CA, USA). The results were analyzed using Flow Jo ver. 10 (Beckton Dickinson, East Rutherford, NJ, USA).

Velazquez-Soto H., Groman-Lupa S., Cruz-Aguilar M., Salazar A.L., Zenteno J.C, & Jimenez-Martinez M.C. (2023). Exogenous CFH Modulates Levels of Pro-Inflammatory Mediators to Prevent Oxidative Damage of Retinal Pigment Epithelial Cells with the At-Risk CFH Y402H Variant. Antioxidants, 12(8), 1540.

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Multiparameter Flow Cytometry Profiling

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The following fluorochrome-conjugated monoclonal antibodies were used in this study: anti-mouse PE/Cy7-conjugated CD45 (Cat# 940335), APC-conjugated Ly6G (Cat# 560599), PE-conjugated CD11b (Cat# 553311), PE-conjugated CD19 (Cat# 553786), and APC/Cy7-conjugated NK1.1(Cat# 560618) (BD Biosciences, San Diego, CA, USA). PerCP/Cy5.5-conjugated F4/80 (Cat# 157317), APC/Cy7-conjugated Ly6C (Cat# 128025), FITC-conjugated CD14 (Cat# 123308), (Biolegend, San Diego, CA, USA), and PE/Cy7-conjugated CD3 (Cat# 25-0031-82) (eBioscience, San Diego, CA, USA). Cells were blocked with FcR blocker (BD Pharmingen) and then incubated with the indicated antibodies under standard protocols. All samples were analyzed using flow cytometry (FACSVerse system, BD Biosciences) with FlowJo (version 7.6.1) software.

Hou X., Liu Q., Gao Y., Yong L., Xie H., Li W., Zhou Y., Liu J., Feng L., Xu L., Shen Y, & Wang H. (2022). Mesencephalic astrocyte-derived neurotrophic factor reprograms macrophages to ameliorate acetaminophen-induced acute liver injury via p38 MAPK pathway. Cell Death & Disease, 13(2), 100.

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Immunophenotyping of Hematopoietic Cells

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For FACS analysis, cells were resuspended in a FACS buffer (PBS with 0.1 % BSA and 2.5 mM EDTA). The cell suspension was then stained with PE-conjugated CD43 (Biolegend, clone MEM-59), APC-conjugated CD34 (BD, clone 581) to detect hematopoietic stem/progenitor cells (HSPC). PE-conjugated CD68 (Biolegend, clone Y1/82A), APC-conjugated CD11b (Biolegend, clone ICRF44), FITC-conjugated CD14 (Biolegend, clone HCD14) were used to detect monocyte/macrophages. Basically, cells were incubated with antibodies for 30 minutes at 4°C, followed with washed and suspended in 0.1% BSA/PBS buffer. PE and APC filters were then used to detect cells double positive for CD43 and CD34 or CD68 and CD11b by signal intensity gating, FITC and APC were used to detect cells double positive for CD14 and CD11b. Negative controls stained with control IgG instead of primary antibodies were always performed with sample measurements. Flowcytometry machine of BD FACSAria II and software of Flowjo were mainly used to collect and analyze the flowcytometry data.

Duan F., Guo L., Yang L., Han Y., Thakur A., Nilsson-Payant B.E., Wang P., Zhang Z., Yan Ma C., Zhou X., Han T., Zhang T., Wang X., Xu D., Duan X., Xiang J., Tse H.F., Liao C., Luo W., Huang F.P., Chen Y.W., Evans T., Schwartz R.E., tenOever B., Ho D.D., Chen S., Na J., Lian Q, & Chen H.J. (2020). Modeling COVID-19 with Human Pluripotent Stem Cell-Derived Cells Reveals Synergistic Effects of Anti-inflammatory Macrophages with ACE2 Inhibition Against SARS-CoV-2. Research Square.

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Immunophenotyping of Dissociated Cells

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Cells were dissociated into single cells with 0.05% trypsin (0.1% EDTA) wherever suitable or rinsed off from culture, and resuspended in a FACS washing buffer (PBS with 5% fetal calf serum (FCS) and 2.5 mM EDTA). The cell suspension was then stained with the desired antibodies. The antibodies used in this study were: CD56 (1:50, clone CMSSB; eBioscience), PE-conjugated CD309 (FLK1, 1:50, clone 7D4–6; Biolegend), PE-conjugated CD13(1:50, clone WM15; BD), FITC-conjugated CD31 (1:50, clone WM59; BD), APC-conjugated CD34 (1:50, clone 581; BD), FITC-conjugated CD43 (1:50, clone MEM-59; Biolegend), PE-conjugated CD43 (1:20, clone eBio84-3C1; eBioscience), FITC-conjugated CD45(1:50, clone 5B1; Miltenyi Biotec), FITC-conjugated CD14 (1:50, clone HCD14; Biolegend), PE-conjugated CD68 (1:50, clone Y1/82A; Biolegend), APC-conjugated CD11b (1:50, clone ICRF44; Biolegend), PE-conjugated CD163 (1:50, clone RM3/1; Biolegend), PE-conjugated CD73 (1:50, clone AD2; Biolegend) and APC/Cy7-conjugated CD163 (1:50, clone 12G5; Biolegend). FITC-conjugated mouse IgG2a (1:20, 130-098-846; Miltenyi Biotec), APC-conjugated mouse IgG1 (1:20, 130-098-877; Miltenyi Biotec) and PE-conjugated mouse IgG1κ (1:20, clone P3.6.2.8.1; eBioscience) were used as isotype-matched negative controls. Data were collected with a FACS Calibur flow cytometer (BD) and analyzed using FlowJo software, version 10.0.7.

Duan F., Huang R., Zhang F., Zhu Y., Wang L., Chen X., Bai L., Guo W., Chang S.C., Hu X, & Na J. (2018). Biphasic modulation of insulin signaling enables highly efficient hematopoietic differentiation from human pluripotent stem cells. Stem Cell Research & Therapy, 9, 205.

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Quantification of Cell Surface Markers

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The expression of cell surface differentiation markers was quantified using flow cytometry. Cells were treated with ZW2-1 (4 μM), and medium with same concentrations of DMSO was used as control. The cells were then washed twice with cold PBS with 0.09% sodium azide and 1% (v/w) bovine serum albumin (BSA) and incubated on ice with antibody conjugated with fluorescein isothiocyanate (FITC conjugated CD11b, FITC conjugated CD14, and PE conjugated CD38; all from Biolegend, Inc., San Diego, CA, USA) in the proportion of 1 : 20, for 30 min. A total of 10,000 cells were analyzed by flow cytometry (FACSCalibur, BD, USA) and the frequency of CD11b-positive, CD14-positive, and CD38-positive cells was determined by using a Flowjo 7.6.1 software.

Wang W., Lv M., Zhao X, & Zhang J. (2015). Developing a Novel Indolocarbazole as Histone Deacetylases Inhibitor against Leukemia Cell Lines. Journal of Analytical Methods in Chemistry, 2015, 675053.

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ACE2 expression in human monocytes

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Negatively selected human monocytes were either untreated (PBS) or treated with IRAK4i (1 µM, PF06650833, Sigma #PZ0327) [23 (link)] for 18 h before staining with FITC conjugated CD14 (Biolegend) and APC labeled ACE2 (R&D Systems) staining for 1 h. Zombie Violet PB450 was used to exclude the dead cells. The gating strategy is shown in Suppl-1.

Umar S., Palasiewicz K., Meyer A., Kumar P., Prabhakar B.S., Volin M.V., Rahat R., Al-Awqati M., Chang H.J., Zomorrodi R.K., Rehman J, & Shahrara S. (2022). Inhibition of IRAK4 dysregulates SARS-CoV-2 spike protein-induced macrophage inflammatory and glycolytic reprogramming. Cellular and Molecular Life Sciences, 79(6), 301.

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