Monocytes were stained using fluorescein isothiocyanate (FITC)conjugated anti-CD45 (BD Biosciences, San Jose, CA, USA), phycoerythrin (PE)-conjugated anti-CD14 (BD Biosciences), peridininÀchloro-phyllÀproteinÀconjugated anti-CD3 (BD Biosciences) and BD Via Probe Cell Viability Solution (7-aminoactinomycin D [7-AAD]; BD Pharmingen, San Diego, CA, USA). PMNs were stained using FITC-conjugated anti-CD45, allophycocyanin (APC)-conjugated anti-CD10 (BD Biosciences) and BD Via Probe Cell Viability Solution (7-AAD). In vitroÀexpanded T lymphocytes were stained using FITC-conjugated anti-CD45, peridininÀchlorophyllÀproteinÀconjugated anti-CD3, APC-conjugated anti-CD4 (BD Biosciences), PE-conjugated anti-CD8 (BD Biosciences) and BD Via Probe Cell Viability Solution (7-AAD). To assess TRAIL receptor expression, monocytes, PMNs and T lymphocytes were tested for PE-conjugated anti-DR4 (BioLegend, San Diego, CA, USA), APC-conjugated anti-DR5 (BioLegend), PE-conjugated anti-DcR1 (BioLegend) and APC-conjugated anti-DcR2 (R&D Systems, Minneapolis, MN, USA). Isotype control antibodies were used for all cell types and antigens analyzed. All samples were acquired using the BD FACSAria III (BD, Franklin Lakes, NJ, USA) flow cytometer and analyzed using the BD FACSDiva software.
Fitc conjugated anti cd45
Manufactured by BD
Sourced in United States
The FITC-conjugated anti-CD45 is a laboratory reagent used in flow cytometry applications. It is a monoclonal antibody that binds specifically to the CD45 cell surface antigen, which is expressed on most human leukocytes. The FITC (fluorescein isothiocyanate) fluorescent label allows for the detection and identification of CD45-positive cells.
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Lab products found in correlation
Pe conjugated anti cd14, by BD (2 mentions)
Adenosine diphosphate (adp), by Merck Group (2 mentions)
Fitc conjugated annexin 5, by Cell Signaling Technology (2 mentions)
Fluorescein isothiocyanate fitc conjugated anti p selectin, by Cell Signaling Technology (2 mentions)
Jon a antibody, by Emfret Analytics (2 mentions)
Phycoerythrin conjugated anti cd69, by BD (2 mentions)
Fitc conjugated anti cd105, by BD (2 mentions)
Fitc conjugated anti cd90, by BD (2 mentions)
Fitc conjugated anti cd34, by BD (2 mentions)
Facsaria 3, by BD (1 mentions)
Via probe cell viability solution, by BD (1 mentions)
Peridinin chloro phyll protein conjugated anti cd3, by BD (1 mentions)
Apc conjugated anti cd4, by BD (1 mentions)
Pe conjugated anti cd8, by BD (1 mentions)
U46619, by Abcam (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Cotinine, by Merck Group (1 mentions)
Nicotine, by Merck Group (1 mentions)
Stir bars, by Chrono-Log (1 mentions)
Thrombin, by Chrono-Log (1 mentions)
10 protocols using fitc conjugated anti cd45
1
Characterizing Immune Cell Phenotypes
2
Platelet Activation Assay with Vaping E-liquid
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Absolute Zero e‐liquid (18 mg nicotine, 30% propylene glycol, 70% vegetable glycerin with a menthol flavor) was obtained from The Vapor Chef (Bristol, PA). ADP, prostacyclin/prostaglandin I2 (PGI2), cotinine methanol solution, and cotinine‐D3 standard were purchased from Sigma Aldrich (St. Louis, MO). U46619 was purchased from Abcam (St. Louis, MO). Fluorescein isothiocyanate (FITC)‐conjugated anti‐P‐selectin and FITC‐conjugated Annexin V were purchased from Cell Signaling Technology, Inc (Danvers, MA). The JON/A antibody was obtained from Emfret analytics (Würzburg, Germany). The phycoerythrin‐conjugated anti‐CD69 and FITC‐conjugated anti‐CD45 were obtained from BD Biosciences (San Jose, CA). Stir bars and other disposables were purchased from Chrono‐Log Corporation (Havertown, PA). Kinetex 1.7 μm EVO C18 100 A‐LC Column 100×2.1 mm, Security Guard ULTRA holder for UHPLC Columns 2.1 to 6.4 mm, and SecurityGuard ULTRA cartridges for EVO‐C18‐UHPLC were purchased from Phenomenex Inc (Torrance, CA). Other reagents were of analytical grade.
3
Platelet Activation and Signaling Pathways
4
Apoptosis Measurement by Flow Cytometry
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Cells were adjusted to a concentration of 10×103 cells/µL using phosphate buffered saline (PBS). A 100 µL aliquot of the cell suspension was placed in a test tube. Cells were stained with 20 µL of fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (BD, Biosciences) for 15 min at room temperature in the dark. Erythrocytes were lysed using 2 mL of an ammonium chloride-based buffered solution. After washing, the cells were adjusted to a concentration of 1×106 cells in 500 µL of the Cytofix/Cytoperm solution (BD, Biosciences) and incubated for 20 min on ice. After centrifugation, cell pellets were washed twice, using 500 µL of Perm/Wash buffer solution (BD, Biosciences), resuspended in 100 µL of Perm/Wash buffer solution, and stained with 20 µL of PE-conjugated caspase-3 for 20 min at room temperature in the dark. After washing and suspending the cells in 500 µL of Perm/Wash buffer solution, cells were analyzed by flow cytometry (FC-500; Beckman Coulter). From each sample, 50,000 events were acquired and analyzed.
5
Flow Cytometry Analysis of MSCs
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MSCs were digested and washed with phosphate-buffered saline (PBS, Boster, Wuhan, China). After incubation with specific antibodies, MSCs were washed again and resuspended in PBS. Flow cytometry was performed using a BD Biosciences Influx Cell Sorter (San Jose, CA, USA). Human phycoerythrin- (PE-) conjugated anti-CD29, fluorescein isothiocyanate- (FITC-) conjugated anti-CD73, FITC-conjugated anti-CD105, FITC-conjugated anti-CD45, PE-conjugated anti-CD14, and PE-conjugated anti-HLA-DR antibodies were used for these experiments (all from BD Biosciences, San Diego, CA, USA).
6
Immunophenotyping of Human BM-MSCs
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Cultured human BM-MSC were harvested with TrypLE Select, and 1 × 106 cells were incubated with primary antibodies for 30 min at 4 °C. The primary antibodies used in this study were FITC-conjugated anti-CD105, PE-conjugated anti-CD73, FITC-conjugated anti-CD90, FITC-conjugated anti-CD45, FITC-conjugated anti-CD34, PE-conjugated anti-CD11b, and PE-conjugated anti-CD14, FITC-conjugated anti-CD19 (BD Bioscience). Cells were washed with blocking reagent and analyzed in a FACS Calibur Flow Cytometer (Beckton Dickinson).
7
Immunophenotyping of Adipose-Derived Stem Cells
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hASCs (P2–P5, 2×106) were tested. For each passage, cells were divided equally into 10 microcentrifuge tubes. Cells in 1 tube served as a negative control, while the remaining cells were incubated with PE-conjugated anti-CD34 (348057; BD Pharmingen, USA), anti-CD73 (550257; BD Pharmingen, USA), anti-CD105 (560839; BD Pharmingen, USA), and IgG1 control (349043; BD Pharmingen, USA), and FITC-conjugated anti-CD45 (347463; BD Pharmingen, USA), anti-CD90 (2296945; BD Pharmingen, USA), anti-CD11a (347983; BD Pharmingen, USA), anti-HLA-DR (347363, BD Pharmingen, USA), or IgG2a control (349051; BD Pharmingen, USA) for 20 min at 4°C before being washed twice with PBS and analyzed by FCM (BD, Heidelberg, Germany).
CCR7-hASCs/GFP-hASCs (P3; GFP-positive in FITC channel) were incubated with PE-conjugated monoclonal antibodies as hASCs and incubated with APC-conjugated anti-HLA-DR (17-9956e; Bioscience, USA).
CCR7-hASCs/GFP-hASCs (P3; GFP-positive in FITC channel) were incubated with PE-conjugated monoclonal antibodies as hASCs and incubated with APC-conjugated anti-HLA-DR (17-9956e; Bioscience, USA).
8
Multiparametric Characterization of Stem Cells
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Cells were trypsinized and resuspended in washing buffer consisting of PBS with 2.5% FBS, and stained with primary antibodies for 30 min at 4 °C. Cells were then stained with streptavidin-PE/Cy5 (1 : 200; BD Pharmingen, Franklin Lakes, NJ, USA) for 30 min at 4 °C in the dark. Primary antibodies used for cell staining were PE-conjugated anti-CD56 (1 : 20; Miltenyi Biotec), biotinylated anti-PDGFRα (2.5 μg/ml; R&D; cat. no. BAF322), FITC-conjugated anti-CD34 (1 : 10; BD Pharmingen), FITC-conjugated anti-CD45 (1 : 10; BD Pharmingen), FITC-conjugated anti-CD90 (1 : 200; BD Pharmingen), FITC-conjugated anti-CD105 (1 : 10; BioLegend, San Diego, CA, USA), and FITC-conjugated anti-CD166 (1 : 20; MBL, Aichi, Japan). Stained cells were analyzed by FACSCalibur or FACSVantage SE (BD Biosciences). Cell sorting was performed on a FACSVantage SE.
9
Immunostaining of Captured Tumor Cells
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Captured tumor cells on the herringbone chips were fixed with 4% paraformaldehyde and subsequently permeabilized with 0.2% Triton X-100 in 1% BSA, all in PBS. Cells were immunostained with phycoerythrin (PE)-conjugated anti-pan cytokeratin (CK) and FITC-conjugated anti-CD45 (BD Biosciences, USA) overnight at 4°C and washed twice with PBS before imaging. Nuclei were stained with DAPI (Sigma-Aldrich, USA).
10
Flow Cytometry Characterization of ASCs
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To confirm the ASC-progenitor phenotype, flow cytometry was performed after 3 days following the protocol in the previous subsection. Briefly, after tissue digestion ASCs (8 × 10 4 ) were washed, resuspended in 1% BSA in PBS, and incubated with FITC-conjugated anti-CD29, APC-conjugated anti-CD31, FITC-conjugated anti-CD44, FITCconjugated anti-CD45, and PE-conjugated anti-CD90 (BD Bioscience, San Jose, CA,USA), respectively, for 1 h at 4°C. Corresponding isotype-matched antibodies were used as controls. FACS analysis was performed using an Accuri C6 cytometer (BD, Franklin Lakes, NJ, USA).
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