Human neutrophil purity, mouse neutrophil counts, and human TLR4 expression were both measured by flow cytometry. Cells were collected and centrifuged at 250 ×g for five minutes. After being resuspended in 100 μL binding buffer for cell purity and counts, human neutrophils were marked with 10 μL PE-conjugated CD15 (BD Biosciences, CA) and 10 μL APC-conjugated CD16 (BD Biosciences), and neutrophil counts were marked with FITC-conjugated CD11b, APC-conjugated Ly-6G and PE-conjugated Ly-6C (BioLegend, CA) for 20 minutes at room temperature. Then, 200 μL binding buffer was added and cells were evaluated by a fluorescence-activated cell sorting (FACS) Calibur device (BD Biosciences). Expression of TLR4 was evaluated by flow cytometry using Goat anti-TLR4 primary antibodies (R&D Systems, USA, #AF1478). Donkey anti-Goat IgG-AlexaFluor 647 (1:500, Absin, China, #abs20027) were used as secondary antibodies. Cells were measured by a FACS Calibur device (BD Biosciences). Cell populations were analyzed using the FlowJo software, v10.4.
Fitc conjugated cd11b
Manufactured by BioLegend
Sourced in United States
FITC-conjugated CD11b is a cell surface marker that binds to the CD11b antigen, which is expressed on the surface of myeloid cells, including monocytes, macrophages, and granulocytes. The FITC (Fluorescein Isothiocyanate) fluorescent label allows for the detection and identification of cells expressing CD11b using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur device, by BD (1 mentions)
Pe conjugated cd15, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (1 mentions)
Fitc conjugated cd14, by BioLegend (1 mentions)
Facscalibur, by BD (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd4 antibody clone gk1, by BioXCell (1 mentions)
Ova257 264 siinfekl, by AnaSpec (1 mentions)
Cell trace red, by Thermo Fisher Scientific (1 mentions)
Celltrace cfse, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Resomer rg502h, by Boehringer Ingelheim (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Polyvinyl alcohol (pva), by Merck Group (1 mentions)
Atto647n, by ATTO-TEC (1 mentions)
α galcer, by Funakoshi (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Attune nxt, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
5 protocols using fitc conjugated cd11b
1
Quantifying Neutrophil Purity and TLR4 Expression
2
Quantification of Cell Surface Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
3
PLGA Nanoparticle Formulation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PLGA (Resomer RG 502 H, lactide/glycolide molar ratio 48:52 to 52:48) was purchased from Boehringer Ingelheim (Germany). Solvents for PLGA preparation (dichloromethane) were obtained from Merck (Germany). Polyvinyl alcohol (PVA) was obtained from Sigma (The Netherlands). R848 was from Enzo Life Sciences poly I:C from Sigma-Aldrich, and endotoxin-free OVA from Hyglos. OVA (257 – 264) SIINFEKL and HPV16 E7(49–57) obtained from Anaspec. α-GalCer was purchased from Funakoshi Co. Ltd. Dimethyl sulfoxide (DMSO) ≥99.9% and Tween 20 was obtained from Sigma (The Netherlands). Atto647N was obtained from Atto-tec GmbH. RPMI 1640 medium (Life Technologies Inc..). PE-conjugated CD3, APC-Cy7 conjugated CD45.2 (BD Biosciences), PerCP conjugated CD8+α (Biolegend), and APC-conjugated H2-Kb/SIINFEKL-Tetramer (Sanquin), PE conjugated CD1d- α-GalCer dextramer (Immudex),FITC conjugated CD11b, PE/Cy7 conjugated CD11c, Alexa488 conjugated F4/80 and NK1.1 (Biolegend), PE/Cy7 conjugated CD154 (Biolegend), Celltrace CFSE, Celltrace- violet and Celltrace red (Life technologies Inc.), were used in flow cytometric cell staining. For the depletion studies, anti-mouse CD4+ antibody clone GK1.5 was obtained from BioXcell. For the detection of CD4+ populations PerCP/Cy5.5 conjugated anti-mouse CD4+ antibody clone RM4-4 was obtained from Biolegend.
4
Gal3 and CD11b Immunophenotyping by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with BD Cytofix/Cytoperm solution (554722, BD Biosciences) for 20 min at 4 °C. After that, samples were washed and permeabilized twice with BD Perm/Wash buffer (554723, BD Biosciences) before sequential labeling with a rabbit anti-Gal3 antibody (1 µg/ml)66 (link), a goat anti-rabbit secondary antibody (1:100, Alexa Fluor 568, A-11011, Invitrogen), and a FITC-conjugated CD11b (1:200, 101205, BioLegend) at 4 °C in the dark for 30 min. The samples were washed three times after each labeling and were resuspended in staining buffer before being subjected to an Attune® NxT acoustic focusing flow cytometer (Life Technologies, USA). Gating strategy used in the study is provided in Supplementary Fig. 15 .
5
Analyzing Immune Cell Infiltrates in Oral Mucosa
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For analysis of immune infiltrate, oral mucosal tissues were harvested and digested in 1 mg/ml collagenase I (Sigma) for 45 min at 37 °C. The digested palate mucosa was filtered through a cell strainer to obtain a single-cell suspension. Single-cell suspensions from palatal samples were stained for live cells using Zombie NIR (Biolegend) dyes in cell-culture grade PBS per manufacturer instructions. Cells were then stained with cell phenotyping antibodies in a 1:1 vol ratio of 3% FBS and Brilliant Stain Buffer (BD Biosciences) according to standard procedures and analyzed on a FACS AriaIIIu flow cytometer (BD Biosciences). The following antibodies were used for cell phenotyping: Zombie NIR Fixable Viability Kit (BioLegend), BV606-conjugated CD45 (BioLegend), PE-conjugated MERTK (BioLegend), PerCP-Cy5.5-conjugated CD64 (BioLegend), APC-conjugated Ly6C (BioLegend), PE-Cy7-conjugated Ly6G (BioLegend), FITC-conjugated CD11b (BioLegend), and APC-Cy7-conjugated CD206 (BioLegend).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!