Platelet suspensions sampled from the experimental and control groups were fixed with 1% phosphate-buffered paraformaldehyde (Sigma-Aldrich, USA) for 20 min and then rinsed with phosphate-buffered saline thrice. The suspensions were then centrifuged at 400 g for 5 min. The platelet concentration was adjusted to 5000/μL. Thereafter, 50 μL of each sample was stained with 5 μL anti-CD62P-FITC (BD Biosciences, USA) and 5 μL anti-CD41 (BD Biosciences) at room temperature for 30 min in the dark, and another 50 μL of each sample was stained with 5 μL mouse IgG1-FITC (BD Biosciences) and 5 μL anti-CD41 under the same conditions as the negative control. The platelets were then washed with 2 mL phosphate-buffered saline and centrifuged at 400 g for 5 min. Finally, the platelets was resuspended in 300 μL 1% phosphate-buffered paraformaldehyde and detected on a FACSCalibur flow cytometer (BD Biosciences) using the CellQuest v3.2 software.
Anti cd41
Manufactured by BD
Sourced in United States
Anti-CD41 is a laboratory reagent used for the identification and enumeration of platelets in biological samples. It is a monoclonal antibody that specifically binds to the CD41 antigen, also known as the GPIIb/IIIa complex, which is expressed on the surface of platelets. The Anti-CD41 reagent can be used in flow cytometry analysis to quantify platelet levels in a sample.
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Lab products found in correlation
Anti cd235a, by BD (2 mentions)
Anti cd45, by BD (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Mouse igg1 fitc, by BD (1 mentions)
Anti cd62p fitc, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Trucount tube, by BD (1 mentions)
Apc labelled annexin 5, by BD (1 mentions)
Confocal microscope, by Leica (1 mentions)
Anti f4 80, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by BD (1 mentions)
Anti ly6g, by BD (1 mentions)
Anti cd62e, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Anti cd34, by BioLegend (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Anti cd11b, by BD (1 mentions)
Facs aria fusion machine, by BD (1 mentions)
Anti ly 6g 6 c, by BD (1 mentions)
Pe conjugated annexin 5, by BioLegend (1 mentions)
9 protocols using anti cd41
1
Flow Cytometric Analysis of Platelet Activation
2
Platelet Depletion via Anti-CD41 Antibody
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Platelets depletion was performed as described previously with penile vein injection of 1 mg/kg low-endotoxin and azide-free anti-CD41 antibody (anti-CD41, BD Biosciences, San Diego, CA), diluted in 200 μL sterile normal saline (0.9% [wt/vol] sodium chloride). Injection of this mixture reduced the number of circulating platelets to <0.05 × 106 value, 1.0–1.2 × 106 published values (23 (link)).
3
Flow Cytometric Analysis of Microparticles
4
Immunostaining Quantification of Liver Tissue
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For the immunostaining assay, the procedures were followed as previously described34 (link). Briefly, fresh liver tissues from APAP treated mice were fixed in 4% paraformaldehyde overnight at 4 °C and then transferred to 20% sucrose in PBS and stored at 4 °C for 24 h followed by mounting liver tissue in O.C.T. and stored at −80 °C. Five-micrometer sections were used for immunostaining. Liver tissue sections were immunostained with the anti-F4/80 (eBioscience, 14-4801-85), anti-VWF (Invitrogen, PA5-16634), anti-CD41 (BD Pharmingen, 553847), or anti-cleaved caspase-3 (Asp175; Cell Signaling, cs9661L) antibody followed by Alexa488 or red-conjugated secondary antibody. Confocal images were obtained using a Leica confocal microscope. The VWF positive areas, CD41 positive areas and number of F4/80 positive cells were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). Briefly, RGB images were converted into 8-bit images, and then a threshold of positive area was adjusted and set up. The positive areas of the images were then analyzed using Analyze > Set Measurements. For counting the number of F4/80 positive cells, Plugins > Analyze > Cell counter were chosen and total cell numbers were manually counted. At least 5 images from each mouse were quantified from four WT mice and five p62 KO mice with 24 h post APAP treatment.
5
Flow Cytometric Quantification of Cell-Derived Microparticles
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Whole blood was obtained by cardiac puncture using heparin as an anticoagulant. Plasma was labeled for MPs of specific cell origin as previously described [21] (link). Forward and side-channels were set to analyze particles using 0.3, 1.1, and 3.0 µm latex beads for calibration (Figure 1A ). The forward and side scatter gate was set based on size and vesicles within the 0.3–1.0 µm were identified as MPs (Figure 1B ) and further analyzed. MPs were then assessed with both source specific markers and Annexin V (BD Pharmingen, San Diego, California, USA) which stains for the presence of phosphatidylserine, a cell surface marker linked to apoptosis [22] (link). Platelet-derived MP levels were assessed using the platelet-specific marker Anti-CD41 (clone: MWReg30, BD Pharmingen, San Diego, California, USA) (Figure 1C ). Neutrophil-derived MP levels were assessed using neutrophil specific marker, Anti-Ly-6G (clone: 1A8, BD Pharmingen, San Diego, California, USA) [23] (link) (Figure 1D ). Endothelial-derived MP levels were assessed using endothelial cell specific marker Anti-CD-62E (clone 10E9.6, BD Pharmingen, San Diego, California, USA) (Figure 1E ). Flow cytometry data acquisition and analysis were performed using a Beckman Coulter Epic flow cytometer with Kaluza software (Indianapolis, IN, USA).
6
Megakaryocyte Differentiation Analysis
7
Isolation of Fetal Liver Cell Populations
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To isolate cells of different stages of differentiation directly from primary fetal livers at E12.5–E13.5, 5–15 fetal livers were pooled together, mechanically dissociated in staining buffer (PBS, 0.2% BSA, 5 mM glucose) and strained through a 30-μM filter. Cells were stained at 4 °C with rabbit IgG (200 μg/ml, Jackson Laboratories) to block Fc receptors. Cells were then incubated with PE-Cy7 conjugated anti-CD71 (RI7217 BioLegend, 1:2000), APC conjugated anti-Ter119 (RUO, BD Biosciences; 1:200), BB700 conjugated CD117 (104D2, BD Biosciences, 1:100), PE-conjugated Annexin V (BioLegend, 1:100), a panel of 5 FITC-conjugated lineage antibodies (anti-CD41, anti-CD45R, anti-CD3e, anti-CD11b, and anti-Ly-6G/6C, all at 1 μg/ml, all BD Biosciences), Cells were then resuspended in FACS running buffer (staining buffer with the addition of 2 mM EDTA). 0.66 μg/ml Hoechst (Invitrogen) was added prior to sorting, which was performed on a BD FACSAria Fusion machine with a 100 μm nozzle. Annexin V was only added for the experiments on the Lrf−/− mice. Single color controls and FMOs were used for calibration.
8
Platelet Depletion in Mice
9
Immunophenotyping of Hematopoietic Progenitors
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The dissociated cells were incubated with antibody madoptixture at 4 °C for 30 min, followed by incubation with 7-AAD antibody at room temperature for 5 min. Cells were sorted and analyzed by flow cytometers FACS Aria II (BD Biosciences) and MoFlo XDP (Beckman Coulter) in the purity model. The FACS data were analyzed with FlowJo software (v10, Tree star). Surface markers for E10.0 early AECs in AGM regions were CD41−CD43−CD45−CD31+CD44+Kit−. Surface markers for E10.0 HECs in AGM regions were CD41−CD43−CD45−CD31+CD44+Kit+CD201+. Surface markers of E11.0 AGM pre-HSCs were CD31+Kit+CD201+. Surface markers for E14.5 fetal liver LT-HSCs were CD45+CD201+CD150+CD48−. Cells were stained using the following antibodies (1:100 diluted): Anti-CD31 (BD, MEC13.3, Catalog# 562939), Anti-CD41 (BD, MWReg30, Catalog# 553848), Anti-CD43 (BD, S7, Catalog# 553270), Anti-CD44 (BioLegend, Catalog# 103044), Anti-CD45 (BD, 30-F11, Catalog# 553079), Anti-CD48 (BioLegend, Catalog# 103432), Anti-CD201 (eBioscience, eBio1560, Catalog# 17-2012-82), Anti-Kit (eBioscience, 2B8, Catalog# 14-1171-82), and Anti-CD150 (BioLegend, Cat# 115904). 7-aminoactinomycin D (7-AAD; eBioscience, Catalog# 00-6993-50) was used to exclude dead cells. The FACS Diva 8 “index sorting” function was activated and sorting was performed in the single-cell mode.
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