Platelet preparation and activation assays were performed according to the methods of Goodall and Appleby45 (link) with minor modifications. Mouse blood was collected under deep anaesthesia in a tube containing 20 unit/ml heparin (Mitsubishi Tanabe Pharma, Osaka, Japan). The whole blood was centrifuged for 5 min at 500 × g to prepare the platelet rich plasma (PRP). The PRP was centrifuged again for 8 min at 300 × g. After the addition of 0.5 μM prostacyclin (Cayman Chemical, Ann Arbor, MI, US), the PRP was centrifuged for 5 min at 1300 × g. The platelet preparation was incubated for 30 min at 37 °C with PBS containing 0.1% bovine serum albumin (Roche Diagnostics, Basel, Switzerland); 2 mM EDTA (Dojindo, Kumamoto, Japan); 0.02 unit/ml apyrase (New England BioLabs, Hertfordshire, UK); and 0.5 μM prostacyclin after washing with the same buffer. The prepared platelet containing fraction was analysed by flow cytometry using Aria II (Becton Dickinson) after staining with anti-CD41 FITC (eBioscience, San Diego, CA, US) and anti-CD62P APC (eBioscience). Platelet activation was evaluated by the reactivity of anti-CD62P APC against the platelet fraction, which was gated with forward/side scatter characteristics and anti-CD41 FITC positive fractions.
Anti cd41 fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Anti-CD41-FITC is a fluorescent-labeled antibody that binds to the CD41 antigen, also known as the platelet glycoprotein IIb (GPIIb). It is commonly used in flow cytometry applications to identify and quantify platelets in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Roche (1 mentions)
Aria 2, by BD (1 mentions)
Prostacyclin, by Cayman Chemical (1 mentions)
Apyrase, by New England Biolabs (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Dojindo Laboratories (1 mentions)
Anti c kit apc, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Streptavidin pe, by Bio-Rad (1 mentions)
Lsr 2 fortessa flow cytometer, by Tree Star (1 mentions)
Anti cd45 pb, by BD (1 mentions)
Sw60ti rotor, by Beckman Coulter (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Kaluza analysis software, by Beckman Coulter (1 mentions)
Anti cd16 fitc, by BD (1 mentions)
Pe conjugated anti p selectin antibody, by Thermo Fisher Scientific (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Anti cd61 pe, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Vav cre mice, by Jackson ImmunoResearch (1 mentions)
5 protocols using anti cd41 fitc
1
Platelet Preparation and Activation Assay
2
Polyploid Megakaryocyte Profiling
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Polyploidy analyses were performed at day 5 of CD9High MEP cultures. Cells were centrifuged at 400 g for 10 minutes, labeled with anti-c-kit-APC (BD Pharmingen), anti-CD41-FITC (eBiosciences) and anti-CD42b-PE (Emfret) antibodies. DNA was stained with Hoechst 33342 (20 μg/mL Eurogentec) during 45 min at 37°C followed by FACS analysis after gating on the Kit-/CD41+/CD42b+ mature megakaryocytic subset from which the percentages of cells displaying different levels of DNA content were recorded.
3
Immunophenotyping and Viability Assays of Blood Cells
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The cells obtained from whole blood were washed twice in PBS with 2% FCS before staining with either anti‐CD11b‐FITC (Abcam) or anti‐CD41‐FITC (eBioscience) with biotinylated anti‐HLL followed by Streptavidin‐PE (Bio‐Rad). Cells were then washed twice before analysis on a BD FACSCALIBUR with CellQuest Pro software. Erythrocytes were identified by forward/side scatter profile.
For viability assays, cells were washed twice with PBS and then incubated with Fixable LIVE/DEAD Near‐IR fluorescent reactive dye (Thermo Fisher Scientific, Paisley, Renfrewshire, UK) for 30 minutes at 4°C. Cells were washed, fixed for 15 minutes in 1% paraformaldehyde, then washed with PBS‐5% FCS and stored at 4°C before acquisition and analysis within 24 hours on an LSRII/Fortessa flow cytometer at the BRC Flow Cytometry Laboratory, King's College London with FlowJo software (Treestar Inc). Macrophages identified by forward/side scatter profile.
For viability assays, cells were washed twice with PBS and then incubated with Fixable LIVE/DEAD Near‐IR fluorescent reactive dye (Thermo Fisher Scientific, Paisley, Renfrewshire, UK) for 30 minutes at 4°C. Cells were washed, fixed for 15 minutes in 1% paraformaldehyde, then washed with PBS‐5% FCS and stored at 4°C before acquisition and analysis within 24 hours on an LSRII/Fortessa flow cytometer at the BRC Flow Cytometry Laboratory, King's College London with FlowJo software (Treestar Inc). Macrophages identified by forward/side scatter profile.
4
Erythrocyte Membrane Proteome Profiling
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Blood collected from 7 donors was washed 4 times in RPMI to remove plasma, platelets, and leukocytes. Residual leukocytes were removed by Plasmodipur filters (Europroxima). Erythrocyte purity was assessed by flow cytometry (Gallios Flow cytometer equipped with 3 lasers: 405, 488, 633 nm; Beckman Coulter). Blood samples were stained with anti-CD45-PB (BD bioscience), anti-CD16-FITC (BD bioscience) to identify granulocytes, and anti-CD61-PE (eBioscence), anti-CD41-FITC (eBioscience) to identify platelets. Data were analyzed by using Kaluza Analysis Software (Beckman Coulter).
RBC membranes obtained by hypotonic lysis (5 mM Na-phosphate, 0.5 mM EDTA, pH 8), were suspended in 0.75 ml of MES-buffered saline (25 mM MES, pH 6.5, 0.15 M NaCl) containing 1% Triton X-100 in ice and homogenized with a potter-elvehjem glass homogenizer. Cell lysates were adjusted to 40% sucrose, overlaid with 1.5 ml of 30% and 1.5 ml of 5% sucrose, and subjected to ultracentrifugation in SW60Ti rotor (Beckman Instruments, 210,000×g, 18 h at 4 °C). 12 fractions (375 µl each) were collected from the top of the gradient. Proteins associated with Triton-insoluble membranes (floating to light density fractions 2–8) were precipitated as described by Wessel and Flugge47 (link) and analyzed by mass spectrometry (Supplemental Methods). Protein identification data are reported in Supplementary data1 .
RBC membranes obtained by hypotonic lysis (5 mM Na-phosphate, 0.5 mM EDTA, pH 8), were suspended in 0.75 ml of MES-buffered saline (25 mM MES, pH 6.5, 0.15 M NaCl) containing 1% Triton X-100 in ice and homogenized with a potter-elvehjem glass homogenizer. Cell lysates were adjusted to 40% sucrose, overlaid with 1.5 ml of 30% and 1.5 ml of 5% sucrose, and subjected to ultracentrifugation in SW60Ti rotor (Beckman Instruments, 210,000×g, 18 h at 4 °C). 12 fractions (375 µl each) were collected from the top of the gradient. Proteins associated with Triton-insoluble membranes (floating to light density fractions 2–8) were precipitated as described by Wessel and Flugge47 (link) and analyzed by mass spectrometry (Supplemental Methods). Protein identification data are reported in Supplementary data
5
Generation and Characterization of Atg7 Knockout Mice
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Suppliers were: phycoerythrin (PE)-labeled JON/A antibody supplied by Emfret (Eibelstadt, Germany); PE-conjugated anti-P-selectin antibody, anti-CD41-FITC, and anti-CD61-PE supplied by eBioscience (San Diego, CA); MitoTracker Green and MitoSox Red supplied by Invitrogen (Carlsbad, CA).
Generation of Atg7f/f;Vav-Cre mice Atg7 Flox/Flox mice (from RIKEN BioResource Center, Ibaraki, Japan) were crossed to Vav-Cre mice (Jackson Laboratory, Sacramento, CA) to obtain Atg7 f/f ;Vav-Cre. Genotyping was performed on tail genomic DNA as described previously [13, 14] . Male and female mice were used equally in all experiments. Each group contained at least six mice. All experiments with animals complied with the institutional protocols on animal welfare and were approved by the Ethics Committee of Soochow University, China.
Generation of Atg7f/f;Vav-Cre mice Atg7 Flox/Flox mice (from RIKEN BioResource Center, Ibaraki, Japan) were crossed to Vav-Cre mice (Jackson Laboratory, Sacramento, CA) to obtain Atg7 f/f ;Vav-Cre. Genotyping was performed on tail genomic DNA as described previously [13, 14] . Male and female mice were used equally in all experiments. Each group contained at least six mice. All experiments with animals complied with the institutional protocols on animal welfare and were approved by the Ethics Committee of Soochow University, China.
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