The presence of CD14, CD25, CD28, CD45, CD61, CD90, TLR1 and TLR4 were analyzed using a flow cytometer (CYTOMICS FC 500, Beckman Coulter, Miami, FL) according to manufacturer's instruction. Anti-CD14-PE-Cy7, CD25-FITC, TLR4-PE, CD28-PE, CD61-PE, CD90-FITC and CD45-APC were purchased from (BD Biosciences) ; anti-TLR1-PE was purchased from (eBioscience).
Cd61 pe
Manufactured by BD
Sourced in United States
The CD61-PE is a fluorescent-labeled antibody that binds to the CD61 antigen, also known as the platelet glycoprotein IIIa (GPIIIa) or integrin beta 3. It is commonly used in flow cytometry applications to identify and quantify platelet cells and platelet-derived microparticles.
Automatically generated - may contain errors
Lab products found in correlation
0.105 m buffered sodium citrate, by BD (3 mentions)
Fibrinogen from human plasma oregon green 488 conjugate, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Corning (3 mentions)
Cellfix, by BD (3 mentions)
Prostaglandin e1, by Merck Group (3 mentions)
Acid citrate dextrose, by BD (3 mentions)
Anti tlr1 pe, by Thermo Fisher Scientific (2 mentions)
Cytomics fc500, by Beckman Coulter (2 mentions)
Cd25 fitc, by BD (2 mentions)
Cd90 fitc, by BD (2 mentions)
Cd45 apc, by BD (2 mentions)
Anti cd14 pe cy7, by BD (2 mentions)
Tlr4 pe, by BD (2 mentions)
Trucount tube, by BD (2 mentions)
Cd62 pe, by BD (2 mentions)
Anti human cd62 pe, by BD (2 mentions)
Cd61 fitc, by BD (2 mentions)
Dihydroethidium dhe, by Merck Group (2 mentions)
Thrombin, by Chrono-Log (2 mentions)
Cd28 pe, by BD (1 mentions)
14 protocols using cd61 pe
1
Multiparametric Flow Cytometry Analysis
2
Blood Platelet Enumeration Protocol
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Each dilution step was done by reverse pipetting to minimize errors. This is performed by depressing the pipette beyond the stop point to draw up additional fluid and then ejecting the sample to the stop point to expel the correct volume. Dilution 1 tube was filled with 190μL (venous) or 180uL (fingerprick) of diluent (PBS 0.1%BSA) whereas dilution 2 and 3 was filled with 150μL of diluent. Before the sample dilutions were prepared, the blood was again mixed completely with a 100uL pipette. Using a positive displacement pipette, 10μL (venous) or 20μL (fingerprick) of blood was pipetted into the dilution 1 containing the 190uL (venous) or 180μL (fingerprick) of diluent. Next 50μL was moved from dilution 1 to dilution 2 tubes then to dilution 3. To three additional 1.5mL tubes, 5μL of both a Coulter CD 41-PE (cat #IM1416U) and BD Biosciences CD 61-PE (cat#561912) was added. Then 100μL of dilution 1–3 blood was added. Samples were mixed on an orbital shaker@1000RPM for 15 minutes at room temperature. Following the incubation, 890μL of diluent was added and pipette mixed. 400 μL of this diluted sample was added to a 5 mL tube with 1600 μL diluent and pipette mixed. This represents a 1:1000 dilution of the initial blood sample.
3
Flow Cytometric Characterization of Extracellular Vesicles
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The labeling and gating of EVs were performed as described by Sáenz-Cuesta and colleagues (28 (link)). Briefly, 4 μl of CD61-PE (Cytgonos) or CD45-PE (BD) monoclonal antibodies were mixed with 40 μl of resuspended EVs and incubated for 20 min. Next, labeled EVs were washed once with 300 μl of filtered PBS, resuspended in further 200 μl of filtered PBS and acquired at low rate in a FACS Canto II flow cytometer (BD). Side and forward scatter were measured on a logarithmic scale with the threshold set at 300 for each parameter to avoid instrument noise (background signal). Then, the lower limit was defined with the exclusion of background noise given by the signal of PBS filtered twice. To define the upper limit of the total MP gate, 1-μm non-labeled polystyrene latex beads were used (Sigma-Aldrich). The events that appeared in this region were included in the total EV count and were further analyzed for specific labeling (positive for PE marker). We defined CD61+ EVs as platelet-derived EVs (PEV) and CD45+ EVs as leukocyte-derived EVs (LEV). The total and cellular origin-specific EV concentrations were obtained using Trucount™ tubes [BD; Ref. (28 (link))].
4
Multiparametric Immune Cell Profiling
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The presence of CD3, CD4, CD14, CD25, CD45, CD61, CD90, TLR1 and TLR4 were analyzed using a flow cytometer (CYTOMICS FC 500, Beckman Coulter, Miami, FL) according to manufacturer's instruction and the data was analyzed using Flow Jo analysis software. Anti-CD14-PE-Cy7, CD25-FITC, TLR4-PE, CD61-PE, CD90-FITC, CD45-APC, CD3-FITC and anti-CD4-PE-Cy5 were purchased from (BD Biosciences) ; anti-TLR1-PE was purchased from (eBioscience).
Cell cycle analysis and apoptosis analysis were performed according to the manufacturer's protocol as previously described 43 (link). All experiments were performed at least in triplicate.
Cell cycle analysis and apoptosis analysis were performed according to the manufacturer's protocol as previously described 43 (link). All experiments were performed at least in triplicate.
5
Isolation and Characterization of Murine Mammary Cells
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Mammary glands were dissected from 10- to 12-week-old female mice at pregnancy day 14.5, cut into pieces, and placed in DMEM/F12 (Gibco) with 5% FBS (Gibco), 300 U/mL collagenase (Gibco) and 100 U/mL hyaluronidase (Sigma) for 2 h at 37°C. After digestion, red blood cells were removed with NH4Cl (STEMCELL Technologies, Vancouver, BC, Canada), and then a single-cell suspension was obtained by sequential dissociation in 37°C preheated 0.25% trypsin (Gibco) for 2 min and in 5 mg/mL Dispase (STEMCELL Technologies) containing 0.1 mg/mL DNase I (Sigma) for 5 min with pipetting, followed by filtration through a 40 μm filter (BD, San Jose, CA, United States). The single-cell suspension was stained with specific antibodies in DPBS containing 2% FBS for 15 min on ice. After being washed three times with DPBS containing 2% FBS, cells were analyzed by FACS Verse or sorted by FACS Aria. The following antibodies were used: CD45-APC (17-0451-82, eBioscience, San Diego, CA, United States), CD31-APC (17-0311-82, eBioscience), TER119-APC (17-5921-82, eBioscience), CD24-PE-Cy7 (25-0242-82, eBioscience), CD29-FITC (11-0291-82, eBioscience), CD61-PE (561910, BD), and Fixable Viability Dye eFluor 450 (65-0863-14, eBioscience). Data analysis was performed with FlowJo 7.6.1.
6
Immunophenotyping and Cell Sorting Protocol
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PCD cultured and SN-cells were harvested by three washes in PBS/5% FCS. AT-cells were detached by vigorous pipetting in PBS/5% FCS and harvested. Cells were stained for the following surface markers using fluorescently labeled antibodies according to manufactures protocol: CD34-alophycocyanin (APC; Miltenyi Biotec) CD41-fluorescein isothiocyanate (FITC; Immunotech), CD51-phycoerythrin (PE; BioLegend) and CD61-PE (BectonDickinson). Corresponding human immunoglobulin G controls were used. At least 104 stained cells were measured by flow cytometry using FACScalibur or LSRII (both BectonDickinson) and were analyzed byFlowJo software version 7.6.5 (Tree Star). Fluorescent activated cell sorting was performed using FACSAria (BectonDickinson) (Fig. 6a ). For dead cell exclusion 4′,6-Diamidin-2-phenylindol (DAPI) was used.
7
Flow Cytometry Antibody Protocol
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Antibodies for flow cytometry (anti-human CD61/FITC, CD61/PE, CD62/PE), Cellfix, and 0.105 M buffered sodium citrate or acid-citrate-dextrose (ACD) were obtained from Becton-Dickinson (Franklin Lakes, NJ, USA). Bovine serum albumin (BSA), tetrahydrofuran (THF), trimethylammonium diphenylhexatriene (TMA-DPH), diphenylhexatriene (DPH), and prostaglandin E1 were purchased from Sigma (St. Louis, MO, USA). Phosphate buffered saline (PBS) was provided from Corning (New York, NY, USA). Fibrinogen from Human Plasma Oregon Green 488 Conjugate was purchased from Invitrogen (Carlsbad, CA, USA). Resolvin E1 was from Cayman Chemical (Ann Arbor, MI, USA). Collagen was obtained from Chrono-log Co (Havertown, PA, USA). All other chemicals, unless otherwise stated, were supplied by Avantor Performance Materials Poland S.A. (Gliwice, Poland).
8
Adenosine Receptor Agonists and Antagonists in Platelet Function
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Adenosine receptor agonists were purchased from Sigma (St. Louis, MO, USA) (NECA (CAS № 35920-39-9)), and Cayman (Ann Arbor, MI, USA) (regadenoson (CAS № 313348-27-5)). LUF5835 (2-amino-6-(1H-imidazol-2-ylmethylsulfanyl)-4-(3-hydroxy-phenyl) pyridine-3,5dicarbonitrile) was synthesized at Laboratory of Molecular Virology and Biological Chemistry, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland. Cangrelor (AR-C69931MX) was from Cayman Chemical (Ann Arbor, MI, USA). Prasugrel metabolite (R-138727) was obtained from BOC Sciences (Shirley, NY, USA). Calcein AM was obtained from Molecular Probes (Eugene, OR, USA). Antibodies anti-human CD61/PErCP, CD61/PE, CD62/PE, PAC-1/FITC, mouse IgG1/PE isotype control, mouse IgG1/FITC isotype control, Cellfix, buffered sodium citrate was purchased from Becton-Dickinson (San Diego, CA, USA). Phosphate buffered saline pH 7.4 (PBS) was obtained from Corning (New York, NY, USA). Dimethyl sulfoxide (DMSO), adenosine diphosphate (ADP), and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). All other chemicals, unless otherwise stated, were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland).
9
Platelet Activation Assay Protocol
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Collagen and thrombin were purchased from Chrono-log Co (Havertown, PA, USA). Antibodies for flow cytometry (anti-human CD62/PE, CD61/PE, CD61/FITC), 0.105 M buffered sodium citrate or acid-citrate-dextrose (ACD), and CellFix were from Becton-Dickinson (Franklin Lakes, NJ, USA). Fibrinogen from Human Plasma Oregon Green 488 Conjugate was received from Invitrogen (Carlsbad, CA, USA). Prostaglandin E1, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), and dihydroethidium (DHE) were provided by Sigma (St. Louis, MO, USA). Phosphate buffered saline (PBS) was delivered by Corning (New York, NY, USA). All other chemicals, unless otherwise stated, were supplied by Avantor Performance Materials Poland S.A. (Gliwice, Poland).
10
Platelet Activation Assay Protocol
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Collagen and thrombin were purchased from Chrono-log Co (Havertown, PA, USA). Antibodies for flow cytometry (anti-human CD62/PE, CD61/PE, CD61/FITC), 0.105 M buffered sodium citrate or acid-citrate-dextrose (ACD), and CellFix were from Becton-Dickinson (Franklin Lakes, NJ, USA). Fibrinogen from Human Plasma Oregon Green 488 Conjugate was received from Invitrogen (Carlsbad, CA, USA). Prostaglandin E1, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), and dihydroethidium (DHE) were provided by Sigma (St. Louis, MO, USA). Phosphate buffered saline (PBS) was delivered by Corning (New York, NY, USA). All other chemicals, unless otherwise stated, were supplied by Avantor Performance Materials Poland S.A. (Gliwice, Poland).
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