BM‐MSCs were characterized just before infusion, by cell surface expression of CD73‐PE, CD90‐PE, CD19‐PE, CD34‐PE, and CD45‐PE (BioLegend, San Diego, CA); CD105‐PE, HLADR‐PE, and CD14‐PE (Becton Dickinson); propidium iodide (PI; Sigma); and isotype IgG1‐PE and isotype IgG2a‐PE (Becton Dickinson). For immunolabeling, cells were incubated for 45 minutes at 2°C–8°C. PI‐negative BM‐MSCs (viable) were characterized using FC500 flow cytometer (Beckman Coulter, Mississauga, ON, Canada) and analyzed using FlowJo (Treestar).
Cd14 pe
Manufactured by BD
Sourced in United States, United Kingdom
CD14-PE is a laboratory reagent that binds to the CD14 receptor on the surface of monocytes and macrophages. It is used for the identification and quantification of these cell types in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 fitc, by BD (15 mentions)
Cd34 pe, by BD (12 mentions)
Facscalibur, by BD (11 mentions)
Cd73 pe, by BD (9 mentions)
Cd105 pe, by BD (8 mentions)
Cd90 pe, by BD (7 mentions)
Hla dr fitc, by BD (7 mentions)
Cd80 fitc, by BD (6 mentions)
Cd33 pe, by BD (6 mentions)
Hla abc fitc, by BD (5 mentions)
Cd19 fitc, by BD (5 mentions)
Cd105 fitc, by BD (5 mentions)
Cd166 pe, by BD (5 mentions)
Facscanto 2, by BD (4 mentions)
Cd83 pe, by BD (4 mentions)
Cd86 fitc, by BD (4 mentions)
Cd4 fitc, by BD (4 mentions)
Cd34 pe cy5, by BD (4 mentions)
Cd34 fitc, by BD (4 mentions)
Cd4 percp cy5, by BD (4 mentions)
78 protocols using cd14 pe
1
Characterization of BM-MSCs Prior to Infusion
2
Urine Immune Cell Composition Analysis
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The 33,494 probes mapped to known genes were used to estimate the immune and kidney cell composition of the samples (Fig. 1 B). The immune response in silico (IRIS) repository of 1,622 genes, classified by their specific expression in multiple immune cell lineages (27 (link)) and previously described transcript sets of 637 genes for kidney-specific cell lineages (28 (link)), was used to estimate the immune and renal cell composition in urine, respectively. Urine samples from 10 random septic patients were analyzed via flow cytometry using an Life Science Research II flow cytometer (Becton Dickinson, Franklin, NJ). Approximately 50–200 mL of urine was collected and processed within 30 minutes of sample collection. The samples were stained with CD3-AF488 (Number 557694; Becton Dickinson), CD4-AF700 (Number 566318; Becton Dickinson), CD8-BV650 (Number 565289; Becton Dickinson), CD14-PE (Number 561707; Becton Dickinson), CD19-APC (Number 561742; Becton Dickinson), and Sytox Blue (Number S34857; Invitrogen; Thermo Fisher Scientific, Waltham, MA).
3
Neutrophil Antigen Expression Analysis
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Granulocytes were examined for abundance of CD14, CD15, CD18 and CD66B neutrophil antigens by ow cytometry analysis. Whole blood samples collected in EDTA were processed within three hours of collection. Fluorescence staining of granulocytes was carried out with CD14-PE, CD66B-FITC, CD18-PerCP and CD15-APC labelled antibodies (Becton Dickinson) directly on whole blood followed by lysis using Optilyse C (Beckman Coulter). 20000 events were acquired on Navioe EX (Beckman Coulter) and data analysis was done using Kaluza® software. Quanti cation of neutrophil antigens in the patient before and during treatment with empagli ozin was compared with normal untreated controls.
4
Comprehensive Immune Cell Phenotyping
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Antibodies used for flow cytometry analysis included: CD2-Tri-Color (TC); CD4-Phycoerythrin (PE) and CD4Allophycocyanin (APC); CD28-Fluorescein Isothiocyanate (FITC); CD8-TC; CD19-APC; CD45RA-APC; CD16-FITC from Life Technologies (Grand Island, NY); CD14-PE; CD27-PE; and IgM-FITC from BD Biosciences (San Jose, CA). Freshly thawed PBMCs from each visit were stained with three to five antibodies: T cells (CD2, CD4, CD8, CD45RA, and CD28); B cells (CD19, IgM, and CD27); NK cells (CD16). The data were collected on a BD FACSCalibur or BD FACSCanto II, and analyzed by Cell-Quest (BD Biosciences) and FlowJo. The gating strategies were presented in Additional file 1 : Figure S1.
5
Monocyte Isolation and Characterization
6
Multiparameter Immune Cell Profiling
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HLA-ABC FITC, HLA-DP,DQ,DR-FITC, CD40 PE, CD80 FITC, CD86 FITC, CD83 PE, CD1a PE, purified anti-human CCR7, CD14 PE, Langerin PE, TLR3 PE were purchased from BD Biosciences (San Jose, CA). CD40 PE, purified rat IgG2a, goat anti-rat IgG PE, mouse IgG1 FITC, mouse IgG1 PE were purchased from Biolegend (San Diego, CA). Recombinant human (rhu)-CCL21 was purchased from R&D Systems (Minneapolis, MN). Rhu-GM-CSF was manufactured by Berlex (Seattle, WA) while rhu-TGFβ1 and rhu-IL-4 were purchased from Biosource (Carlsbad, CA). Poly-ICR was provided by Nventa Biopharmaceuticals/Akela Pharma (Austin, TX). Poly-ICLC was provided by Oncovir, Inc. (Washington, DC) HPV16L1L2 virus-like particles (VLP) and chimeric HPV16L1L2-E7 VLP (HPV16 cVLP) were produced in insect cells and purified as previously described [28] (link). Endotoxin levels in VLP preparations were found to be below 0.06 EU using an E-toxate kit (Sigma-Aldrich).
7
Flow Cytometry Protocol for Cell Characterization
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Flow cytometry was performed as described previously46 (link). Briefly, cells were resuspended in PBS and incubated for 30 min at 4 °C with antibodies as indicated below. After staining, the cells were analyzed on a flow cytometer (BD FACS Canto II, BD Biosciences, CA, USA). The following antibodies were used: PECAM1-PE (130-092-653, Miltenyi Biotec, CA, USA), CD14-PE (555398, BD Biosciences, CA, USA), CD11b-PE (555388, BD Biosciences, CA, USA), CD34-PE (555822, BD Biosciences, CA, USA), KDR-PE (FAB357P, R&D Systems, MN, USA), CD3-FITC (555916, BD Biosciences, CA, USA), CD4-FITC (555346, BD Biosciences, CA, USA), CD19-FITC (555412, BD Biosciences, CA, USA), CD15-FITC (555401, BD Biosciences, CA, USA) and appropriate isotype control antibodies. Flow cytometric data were analyzed with FlowJo (Tree Star, Inc., Ashland, OR, USA) using appropriate isotype-matched controls.
8
Characterizing Human Dental Pulp Stem Cells
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The cell surface antigen expressions of the third-passage hDPSCs were analyzed after the cells were incubated with antibodies for human CD73 phycoerytrin (PE), CD90 PE, CD146 fluorescein isothiocyanate (FITC), CD29 allophycocyanin (APC), CD105 PE, CD45 FITC, CD34 PE, CD14 PE, CD25 APC, and CD28 PE (BD Biosciences, CA, USA) at room temperature in the dark.
The control antibodies were phycoerythrin-conjugated or fluorescein isothiocyanate-conjugated and allophycocyanin-conjugated Mouse IgG1 and Mouse IgG2 (BD Biosciences, San Diego, CA, USA). The flow cytometry outcomes were examined using a flow cytometer (BD, FACSCalibur, San Jose, CA, USA).
The control antibodies were phycoerythrin-conjugated or fluorescein isothiocyanate-conjugated and allophycocyanin-conjugated Mouse IgG1 and Mouse IgG2 (BD Biosciences, San Diego, CA, USA). The flow cytometry outcomes were examined using a flow cytometer (BD, FACSCalibur, San Jose, CA, USA).
9
Isolation of CD34+ Hematopoietic Stem Cells
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Bone marrow samples were harvested from human subjects from the posterior iliac crest using a Yamshidi needle, with aspirations at 5–7 different levels of approximately 10 ml at each level29 (link). The study has been approved by the Ethics Committee for Human Subjects at the University Heidelberg and written informed consent was obtained from each individual. We recruited 17 subjects with the age ranging from 21 to 71 years for the different experiments within this study. The bone marrow aspirates were processed by FICOLL density fractionation for isolation of mononuclear cells (MNCs). After staining with CD34-APC and CD14-PE (both from BD Biosciences, San Jose, CA) the CD34+ cell population was isolated using a FACSAria II flow cytometry cell sorter (BD Biosciences). After Fluorescence Activated Cell Sorting (FACS), the sorted cells were analyzed for their purity by reanalyzing an aliquot of the respective cell population10 (link).
10
Phenotypic Characterization of T-cell Subsets
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MSCs were analyzed using the following antibodies: CD73-PE, CD105-PE, CD14-PE, CD45-FITC, CD90-APC (BD Biosciences), CD106-Pe-Cy5, CD34-Pe-Cy5 (BD Biosciences), HLA-DR-Qdot-605 (Invitrogen), mouse mAb specific to FAP (eBioscience) and PE-conjugated anti-mouse (BD Biosciences). Isotype-matched, fluorochrome-conjugated, mAb were used as negative controls.
Cultured SCS were stained, data acquired and analyzed as previously described using the following antibodies [11] (link); Surface: CD3-Qdot-605, CD4-Qdot-655 (Invitrogen), CXCR5-Alexa-488, CD25-APC-Cy7, PD-1-Brilliant Violet-421; Intracellular: Bcl-6-PE-CF594, active caspase-3-PE (BD Biosciences), and FoxP3-PE-Cy7 (eBioscience).
FL T-cell subsets were flow cytometrically sorted using the following markers; T-cell: DAPI−CD3+CD4+CD19−; TFH: Propidium iodide−CD3+CD4+CXCR5+PD-1+CD25−. Following the sort, an aliquot of TFH were permeabilized and stained with Bcl-6 and FoxP3 to confirm the TFH phenotype. TFH cells were defined as CD3+CD4+CXCR5+PD-1+CD25−Bcl-6+; TFR were defined as CD3+CD4+CXCR5+PD-1+CD25+Bcl-6+FoxP3+ and Tregs were defined as CD3+CD4+CD25+FoxP3+.
Cultured SCS were stained, data acquired and analyzed as previously described using the following antibodies [11] (link); Surface: CD3-Qdot-605, CD4-Qdot-655 (Invitrogen), CXCR5-Alexa-488, CD25-APC-Cy7, PD-1-Brilliant Violet-421; Intracellular: Bcl-6-PE-CF594, active caspase-3-PE (BD Biosciences), and FoxP3-PE-Cy7 (eBioscience).
FL T-cell subsets were flow cytometrically sorted using the following markers; T-cell: DAPI−CD3+CD4+CD19−; TFH: Propidium iodide−CD3+CD4+CXCR5+PD-1+CD25−. Following the sort, an aliquot of TFH were permeabilized and stained with Bcl-6 and FoxP3 to confirm the TFH phenotype. TFH cells were defined as CD3+CD4+CXCR5+PD-1+CD25−Bcl-6+; TFR were defined as CD3+CD4+CXCR5+PD-1+CD25+Bcl-6+FoxP3+ and Tregs were defined as CD3+CD4+CD25+FoxP3+.
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