Expression of cell surface molecules of canine AT-MSCs at passage 3 was evaluated by flow cytometry. Briefly, cells were harvested from cell flasks by enzymatic digestion with 0.25% trypsin-EDTA (Sigma-Aldrich). Next, cells were stained with conjugated antibodies (anti-CD45-Alexa 647, anti-CD19-FITC, anti-CD14-PE, anti-CD73-APC, anti-CD90-APC, anti-CD105-PE, and anti-CD44-FITC; all from eBioscience, San Diego, CA, USA) diluted in PBS containing 0.5% BSA. AT-MSCs were incubated for 20 min in the dark at 4°C. Following incubation, cells were washed with PBS and centrifuged at 300 ×g for 5 min. Cell pellets were resuspended with PBS for data acquisition using a BD FACSAria™ II flow cytometer. Data were analyzed using FlowJo software (FlowJo LCC, Ashland, OR, USA).
Anti cd14 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-CD14-PE is a fluorescently-labeled monoclonal antibody that specifically binds to the CD14 cell surface receptor. CD14 is a marker expressed on the surface of monocytes and macrophages. This product can be used in flow cytometry applications to identify and quantify CD14-positive cell populations.
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Lab products found in correlation
Facsaria 2 flow cytometer, by BD (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Anti cd44 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd105 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd19 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd73 apc, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd16 cd32, by BD (1 mentions)
Accuri flow cytometer, by BD (1 mentions)
Staining buffer, by R&D Systems (1 mentions)
Anti cd3 apc, by BD (1 mentions)
Anti cd45 fitc, by BD (1 mentions)
Anti cd3 fitc, by Thermo Fisher Scientific (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Penicillin and streptomycin, by Solarbio (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Anti cd86 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd11c fitc, by Thermo Fisher Scientific (1 mentions)
Anti hla dr fitc, by Thermo Fisher Scientific (1 mentions)
Anti dc sign antibody, by R&D Systems (1 mentions)
8 protocols using anti cd14 pe
1
Phenotypic Characterization of Canine AT-MSCs
2
Flow Cytometric Analysis of LPS-Cell Interactions
3
Immune Cell Characterization of Stromal Vascular Fraction
4
Isolation of T Cells and Macrophages from Mouse Spleen
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The spleens from C57BL/6 mice were minced and fibrous material was removed by filtration through a 70 μm cell strainer (BD Falcon). Erythrocytes were lysed with NH4Cl solution (Tiangen) and T cells were purified on nylon wool columns (Ye et al., 2008 (link)), and followed by negative selection using MagCellect Mouse CD3+ T Cell Isolation Kits (R&D Systems). Cells were collected and stained with anti-CD3-FITC (CAT#85-11-0032-82, eBioscience) at 4°C for 30 min and analyzed by flow cytometry. For isolation of peritoneal macrophages, mice were injected intraperitoneally with RPMI 1640 (HyClone), and cells were harvested from the peritoneal cavity 10 min after injection. The cells were washed with phosphate-buffered saline (PBS), plated, and allowed to adhere at 37°C for 2 h in Dulbecco's Modified Eagle's Medium (DMEM; HyClone). At the end of that period, non-adherent cells were removed and macrophages were harvested. The purity of macrophages was determined by flow cytometry using anti-CD14-PE (CAT#12-0141, eBioscience).
5
Isolation and Characterization of Rheumatoid Arthritis Synoviocytes
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Synoviocytes were isolated from synovial tissue specimens that were obtained from patients with RA undergoing total joint replacement surgery. Using enzymatic digestion method, the tissue samples were minced into 1-2 mm3 pieces and treated with 2.5 mg/ml type I collagenase (Gibco, USA) in Dulbecco's modified Eagle's medium (DMEM) for 2–4 h at 37°C with 5% CO2. Dissociated cells were centrifuged for 5 min at 300g, and cell pellet was resuspended in DMEM +10% fetal calf serum (FCS) (Gibco, USA), 2 mM L-glutamine, and 100 IU/ml penicillin and streptomycin (Solarbio Life Sciences, China) and seeded in 75 cm2 flasks and incubated overnight. Synoviocytes from passages 4–6 were used in each experiment. The cells were morphologically homogeneous and exhibited the appearance of synovial fibroblasts. The purity of cells was tested by flow cytometry using anti-CD14-PE (eBioscience, USA), anti-CD68-APC (Miltenyi Biotec, Germany), and anti-vimentin-ALEXA 488 (BD, USA), and 99% of isolated cells was FLS (CD14− CD68− vimentin+).
6
Multiparametric Flow Cytometry Analysis
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Anti-CD11c-FITC, anti-HLA-DR-FITC, anti-CD-86-PE, anti-DC-SIGN-PE, and anti-CD14-PE were purchased for flow cytometry from eBioscience (San Diego, CA, USA). Anti-DC-SIGN antibody used to block the DC-SIGN receptor was from R&D system (Minneapolis, MN, USA). Anti-ANXA2 antibody was from R&D system or GeneTex (Irvine, CA, USA). Alkaline phosphatase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were from Bioscience (Taipei, Taiwan, ROC). Human IL-10 and IL-12 antibodies were from PeproTech. Rabbit IgG (H+L) reabsorbed secondary antibody (conjugated DyLight 594) and anti-human IgG (Fc fragment) antibody (conjugated FITC or HRP) were from GeneTex.
7
Monocyte Apoptosis Analysis by Flow Cytometry
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Apoptosis of CD14+ monocyte was assessed using Southern Biotech Apo Screen Annexin V apoptosis detection kit (Annexin V-FITC, 7-AAD solution and Annexin V binding buffer). This assay involves staining peripheral blood mononuclear cells with Annexin V-FITC (a phospholipid-binding protein binding to disrupted cell membranes) in combination with 7-AAD (a vital dye binding to DNA penetrating into apoptotic cells). Fluorescence-activated cell sorting (FACS) analysis of CD14+ monocytes that are in early (annexin V+/7-AAD−) or late (annexin V+/7-AAD+) apoptotic phase was performed. The percentage of apoptotic CD14+ monocytes (out of total circulating CD14+ cells) was assessed by staining peripheral blood mononuclear cells for 3 color FACS analysis employing anti-CD14-PE (eBioscience), Annexin V-FITC and 7-AAD (SouthernBiotech). The cells were then washed again with PBS and re-suspended in 100 µL Annexin V-FITC binding buffer and incubated with 5 µL of Annexin V-FITC for 15 min at room temperature. Another 200 µL of binding buffer and 5 µL of 7-AAD solution were added without washing, and 100,000 cells were acquired by flow cytometry (FACS Calibur, Becton Dickinson) and analyzed by CellQuest software (BD Bioscience). All analyses and readings were made by researchers who were blinded to the study questions.
8
Quantification of Platelet-Leukocyte Complexes
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Platelet-leukocyte complexes (PLC) are defined as monocytes, granulocytes or lymphocytes that are bound to platelets. Citrate-anticoagulated whole blood was collected prior to and 1 h after the transfusion, and within 5 min after collection the blood was added to tubes containing Hepes and antibodies. The PLC were measured by using a platelet marker (anti-CD61-PerCP; BD Bioscience) in combination with a marker for monocytes (anti-CD14-PE; eBioscience, San Diego, CA), granulocytes (anti-CD66b-FITC; Beckman Coulter) or lymphocytes (anti-CD4-PE; eBioscience). After gentle mixing and incubation for 30 min at room temperature in the dark, the samples (total volume 55 μL) were fixed with 0.5 mL 0.3% paraformaldehyde-containing Hepes buffer. After 60 min of fixation, red blood cells were lysed by addition of distilled water [35] and samples were measured by flow cytometry. Leukocytes were identified by the characteristic side scatter and the pan leukocyte marker (anti-CD45-APC; BD Bioscience). Samples were analyzed by percentage CD61 positive cells within the population of monocytes, granulocytes and lymphocytes. The threshold of platelet-leukocyte binding was set at 1% of the isotype control.
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