Frozen PBMC from the 8 APECED patients and 10 healthy donors were used for measuring STAT1 mRNA expression by quantitative PCR (qPCR). To determine the percent of live CD14+ monocytes within PBMC, an aliquot of the PBMC was incubated with LIVE/DEAD® Fixable Violet Dead Cell Stain Kit (ThermoFisher) in 4°C followed by staining with FITC-conjugated anti-human CD14 (Becton Dickinson cat# 555397). Data were collected with LSRII (Becton Dickinson) and analyzed with FlowJo software (Treestar, Ashland, OR, USA). Among the live PBMC, the mean percentage of CD14+ monocytes was similar in the patient and healthy donor groups: 7.2 ± 0.8 vs. 7.7 ± 1.7, respectively (p = 0.75). For mRNA extraction, the RNeasy kit (Qiagen) was used, according to the manufacturer’s instructions. To convert mRNA to cDNA, the high-capacity cDNA reverse transcription kit (Applied Biosystems) was used. qPCR was then performed with TaqMan detection (TaqMan® Universal Master Mix II, with UNG; ThermoFisher), using the 7,500 real-time PCR system (Applied Biosystems) and predesigned primer and probe mixes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ThermoFisher) or STAT1 (ThermoFisher). All qPCR assays were performed in duplicate and results were normalized to GAPDH transcript levels using the threshold cycle (Ct) method.
Fitc conjugated anti human cd14
Manufactured by BD
FITC-conjugated anti-human CD14 is a fluorescently labeled monoclonal antibody that binds to the CD14 antigen on the surface of human cells. CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein that acts as a co-receptor for the detection of lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls. This antibody can be used to identify and enumerate CD14-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facs lysing solution, by BD (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Live dead fixable violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix 2 with ung, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti human foxp3, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti human cd56, by BD (1 mentions)
Brilliant violet bv421 conjugated anti human pd l1, by BD (1 mentions)
Bv510 conjugated anti human cd3, by BD (1 mentions)
Fluorescein isothiocyanate fitc conjugated anti human cd4, by BD (1 mentions)
Phycoerythrin pe conjugated anti human pd 1, by Beckman Coulter (1 mentions)
Pe conjugated anti human gal 9, by BioLegend (1 mentions)
Peridinin chlorophyll protein percp conjugated anti human cd56, by BD (1 mentions)
Allophycocyanin apc conjugated anti human tim 3, by R&D Systems (1 mentions)
Apc h7 conjugated anti human cd8, by BD (1 mentions)
8 protocols using fitc conjugated anti human cd14
1
Quantifying STAT1 mRNA in APECED PBMC
2
Monocyte Subset Identification by Flow Cytometry
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Monocyte subsets were defined based on surface expression of CD14 and CD16 [12] (link), [13] (link). Briefly, whole blood samples (25 μL) anticoagulated with K3EDTA were incubated with the saturating concentration of PE-conjugated mouse antihuman CD16 and FITC-conjugated antihuman CD14 (Becton Dickinson) for 30 minutes in the darkness. The samples were lysed with RBC lysing solution (Becton Dickinson), centrifuged for 5 minutes at 350 ×g and resuspended in PBS. Approximately 10,000 events were acquired in a flow cytometer (Becton Dickinson) and monocyte cell populations were selectively gated based on their forward-scattered light and side-scattered light. Cell isotype control antibodies were used to define background levels. Percentages of CD14+CD16− (classical), CD14+16+ (intermediate), and CD14dimCD16+ (nonclassical) were calculated from the dot plots using statistical package of the Cell Quest software (Becton Dickinson). Isotype matched PE- and FITC-conjugated mouse IgG served as controls for nonspecific staining.
3
Measuring IFN-γ Receptor Expression in APECED
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Frozen PBMC from eight APECED patients and eight healthy donors were used for measuring IFN-γ receptors 1 and 2 levels on CD14+ monocytes. Cells were resuspended at 106 cells per 100 µl in PBS and incubated with LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (ThermoFisher) in 4°C followed by staining with FITC-conjugated anti-human CD14 (Becton Dickinson cat# 555397), PE-conjugated anti CD119 (IFN-γ receptor 1; IFN-γR1; Becton Dickinson cat# 558937), or APC-conjugated anti-IFN-γ receptor 2 (IFN-γR2; R&D cat# FAB773A) for 30 min. PE-conjugated IgG2b κ isotype control (Cat# 555058) and APC-conjugated IgG isotype control (R&D cat# IC108A) were used for control staining. Cells were then washed with PBS/2%FBS and fixed with 1% PFA. Data were collected with LSRII (Becton Dickinson) and analyzed with FlowJo software (Treestar, Ashland, OR, USA). The geometric mean fluorescence intensity on monocytes for each receptor was calculated after subtracting the geometric mean fluorescence intensity of the corresponding isotype control staining.
4
Flow Cytometric Immunophenotyping of PBMCs
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Freshly thawed PBMC were used for surface and intracellular staining regarding flow cytometric analysis. The following monoclonal antibodies were used: Brilliant Violet (BV421)-conjugated anti-human PD-L1 (BD Biosciences), BV510-conjugated anti-human CD3 (BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated anti-human CD4 (BD Biosciences), FITC-conjugated anti-human CD14 (BD Biosciences), (phycoerythrin (PE)-conjugated anti-human PD-1 (Beckmann-Coulter), PE-conjugated anti-human Gal-9 (Biolegend), Peridinin Chlorophyll Protein (PerCP)-conjugated anti-human CD56 (BD Biosciences), PerCP-conjugated anti-human Perforin (BD Biosciences), allophycocyanin (APC)-conjugated anti-human TIM-3 (R&D Systems), APC-conjugated anti-human CD56 (BD Biosciences), APC-conjugated anti-human FoxP3 (eBioscience), APC/H7-conjugated anti-human CD8 (BD Biosciences).
5
Monocyte Subsets Identification and Analysis
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The enriched monocytes were thawed and suspended in PBS supplemented with 10% fetal bovine serum (Gibco, Invitrogen), and then stained with phycoerythrin [PE]-conjugated anti-human CD14 and fluorescein isothiocyanate [FITC]-conjugated anti-human CD16 or their isotype controls which were PE and FITC-conjugated mouse IgG1, respectively (κ isotype control; BioLegend), for identifying monocytes before scRNA-seq. Fluorescence intensity was examined by a Beckman Gallios Flow Cytometer, and analyzed by flow cytometry software Kaluza analysis 2.0 (Beckman Coulter Life Sciences). For the validation of SELL+ CM in KD infants, the mononuclear cells were thawed, and stained with FITC conjugated anti-human CD14 (BD Pharmingen™), PE-Cy7 conjugated anti-human CD16 (BD Pharmingen™), PE-conjugated SELL (BioLegend), or their isotype controls (BioLegend). Fluorescence intensity was achieved on a Beckman Gallios Flow Cytometer, and analyzed by flow cytometry software FLOWJo (BD Pharmingen™). Student’s t test was used to compare the ratio of SELL+CM to CM between the healthy and KD infants.
6
Cytokine Production in Monocytes after TLR2 Stimulation
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Whole blood cells collected in sodium heparin tubes were polyclonally stimulated for 18 h with specific agonists for human TLR2 (HKLM) and TLR2/TLR6 heterodimer (FSL-1) (InvivoGen, San Diego, CA, USA) in the presence or absence of brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) in polypropylene tubes, as previously described [25 (link)]. After culture, cells were stained with FITC-conjugated anti-human CD14 (BD Biosciences) to identify the monocyte population. Then, red blood cells were lysed with FACS lysing solution (BD Biosciences) for 10 min. After being washed, the cells were permeabilised and intracellularly stained with phycoerythrin-labelled monoclonal antibodies against cytokines: interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6; and analyzed by flow cytometry using FACSDiva Software.
7
Neutrophil ERV1 Expression in Type 2 Diabetes
8
Assessing TLR8-Mediated Cytokine Production
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TLR8 activity was assessed by measuring the production of intracellular cytokines in monocytes as previously described [26] . Briefly, blood cells were stimulated with the TLR8 agonist ssRNA40 (InvivoGen, San Diego, CA) for 18 h in the presence of brefeldin A (Sigma-Aldrich, St Louis, MO) to prevent cytokine release. Cells were then stained with FITC-conjugated anti-human CD14 (BD Biosciences, San Jose, CA) to identify the monocyte population. Erythrocytes were lysed with FACS lysing solution (BD Biosciences). Mononuclear cells were permeabilized and intracellularly stained with phycoerythrin (PE)-labelled monoclonal antibodies against IL-1, TNF or IL-6 (BD Biosciences). Cytokine expression was analyzed by flow cytometry using Cell Quest Pro Software (BD Biosciences).
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