The following antibodies from eBioscience were used for FCM: FITC-anti-mouse-CD41, APC-anti-mouse-CD42, PE-anti-mouse-CD71, PE-anti-human-CD34, APC-anti-human-CD61, FITC-anti-mouse-CD34 and PE/FITC- anti-human-CD41a. A rat anti-mouse-CD41 monoclonal antibody (Abcam) diluted 1:50 in PBS was used for immunohistochemical staining. CD63 mouse monoclonal antibody (Santa Cruz Biotechnology), RHOB monoclonal antibody (ABclonal) and GAPDH monoclonal antibody (Cell Signaling Technology) were diluted 1:1000 and used for Western blotting, and 1 μg per mL human TPO antibody or human TPO receptor antibody (R&D Systems) was used to neutralize TPO in PMPs. The information on the antibodies used in our study is provided in Supplementary Table 2 .
Fitc anti mouse cd41
Manufactured by Thermo Fisher Scientific
FITC-anti-mouse-CD41 is a fluorescently labeled antibody that binds to the CD41 antigen expressed on mouse platelets. It is designed for use in flow cytometry applications to identify and quantify mouse platelet populations.
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Lab products found in correlation
Gapdh monoclonal antibody, by Cell Signaling Technology (1 mentions)
Anti mouse cd71 pe, by Thermo Fisher Scientific (1 mentions)
Fitc anti mouse cd34, by Thermo Fisher Scientific (1 mentions)
Cd63 mouse monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Streptavidin pe, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Lumi aggregometer, by Chrono-Log (1 mentions)
Aggro link software, by Chrono-Log (1 mentions)
Thrombin, by Merck Group (1 mentions)
Alexa fluor 405, by Thermo Fisher Scientific (1 mentions)
Anti vwf antibody, by Merck Group (1 mentions)
Methocult media, by STEMCELL (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Adenosine diphosphate (adp), by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
3 protocols using fitc anti mouse cd41
1
Platelet Characterization Antibody Panel
2
Platelet Aggregation and Turnover Assay
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Platelet-rich plasma was isolated as previously described (14 (link)). Platelets used for analyses were isolated from mice fed the chow diet for 20 weeks. Platelet count was adjusted with HEPES-Tyrode’s solution to have a final concentration of 2 × 105 platelets/μl. Platelet aggregation was measured by a Lumi-aggregometer and AggroLink software (Chrono-Log, Havertown, PA). Samples of 0.25 ml were stimulated with ADP (2.5 μM) for 5 min while stirring at 37°C and light transmission was measured over time.
For the determination of platelet turnover, platelets were labeled in vivo by injecting 200 μl of 3 mg/ml NHS-biotin (Pierce; EZ-Link). Every 24 h for 4 days, 5 μl of blood were collected from the tail vein into 125 μl of HEPES/Tyrode’s solution supplemented with anticoagulants (85 mM sodium citrate, 69 mM citric acid) and 20 mg/ml glucose. Samples were incubated with FITC anti-mouse Cd41 (eBioscience) and PE-streptavidin (BD Pharmingen) for 30 min. In these studies, 50,000 events were analyzed at BD FACSCalibur flow cytometer.
For the determination of platelet turnover, platelets were labeled in vivo by injecting 200 μl of 3 mg/ml NHS-biotin (Pierce; EZ-Link). Every 24 h for 4 days, 5 μl of blood were collected from the tail vein into 125 μl of HEPES/Tyrode’s solution supplemented with anticoagulants (85 mM sodium citrate, 69 mM citric acid) and 20 mg/ml glucose. Samples were incubated with FITC anti-mouse Cd41 (eBioscience) and PE-streptavidin (BD Pharmingen) for 30 min. In these studies, 50,000 events were analyzed at BD FACSCalibur flow cytometer.
3
Immunophenotyping of Mouse Hematopoietic Stem Cells
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FITC-anti–mouse CD41, FITC-anti–human CD41, anti–mouse CD127, anti–mouse CD34, and anti–mouse CD41, FITC-anti–mouse c-Kit, APC-anti–mouse Sca-1, PE-CY7-anti–mouse CD16/32 antibodies, and eFluor450 lineage detection cocktail were purchased from eBioscience. PercP-CY5.5 goat anti–rat IgG and the antibodies against myc-tag, Mad2, CCP6, and GST-tag were purchased from Santa Cruz Biotechnology, Inc. The antibodies against Flag-tag, β-actin, and GFP-tag were from Sigma-Aldrich. Anti-VWF antibody was purchased from Millipore. GT335 (anti-glutamylation) antibody was from AdipoGen. Antibody against TTLL6 was from Novus Biologicals. The antibodies against Aurora B, Aurora A, pThr288 of Aurora A, tubulin, and CD42 were from Bioss. Anti-pThr232 antibody of Aurora B was from Antibodies Online. Alexa Fluor 405–, Alexa Fluor 488–, Alexa Fluor 594–, and Alexa Fluor 649–labeled secondary antibodies were from Molecular Probes. ADP, Thrombin, collagen, Hoechst 33342, and phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma-Aldrich. TPO was from PeproTech. Aurora B kinase inhibitor AZD-1152 was from Selleck Chemicals. Methocult media were from STEMCELL Technologies.
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