Whole blood or washed platelets were labeled with anti-human BV421 CD62p (P-selectin; 1:100, AK4; 304926; BioLegend), BV421 CD42b (1:300, HIP1, 303930; BioLegend), fluorescein isothiocyanate (FITC) CD61 (β3; 1:25, RUU-PL7F12; 348093; BD), TO (200 ng/mL; 390062; Sigma), phycoerythrin (PE) CD42b (GPIbα 1:50, HIP1, 555437; BD), PE CD62p (1:100, AK4; 304906; BioLegend), AF599 Mitotracker Ros CMX (mitochondrial dye 5 μM; 9082; Cell Signaling), allophycocyanin (APC) CD62p (1:100, AK4; 304910; BioLegend), and/or APC HLA I (1:100, W6/32; 311410; BioLegend); or anti-mouse BV421-CD62p (1:100, VI P-44; 304926; BioLegend), FITC-CD41a (1:100, MWReg30; 133904; BioLegend), FITC-conjugated streptavidin (1:10, 405201; BioLegend), BD Retic-count (349204; BD), and/or APC-CD41 (1:100; MWReg30; 17-0411-82; ThermoFisher). A polyclonal rat anti-mouse GPIb antibody (R300; Emfret) was used to induce thrombocytopenia in mice. Isotypes BV421 mouse IgG1 (1:100; MOPC-21; 400157; BioLegend), BV421 rat IgG1(1:100, A110-1; 562604; BD), PE or APC mouse IgG1 (1:100; MOPC-21; 400112 and 400120, respectively; BioLegend) were used to quantify CD62p exposure. Fluorescence Minus One was used for all other markers. AF674 SiR tubulin probe (4 μM; CY-SC002; SpiroChrome) was used for ISFC tubulin labeling.
A110 1
Manufactured by BD
Sourced in United States
The A110-1 is a laboratory centrifuge designed for general-purpose applications. It can be used to separate components of a liquid mixture based on their density differences. The centrifuge provides a maximum speed of 4,000 RPM and a maximum RCF (Relative Centrifugal Force) of 2,000 x g. The unit dimensions are approximately 12 x 14 x 9 inches and it has a weight of around 20 pounds.
Automatically generated - may contain errors
3 protocols using a110 1
1
Multicolor Flow Cytometry of Platelets
2
Murine Hepatic Ischemia-Reperfusion Injury
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Partial (70%) warm hepatic I/R in mice was performed as previously described (27 (link)). After 60 min of liver ischemia by vascular occlusion, the clip was carefully removed, and mice were reperfused and euthanized immediately at 0, 6, 12, or 24 h post-reperfusion. Blood and liver tissue samples were collected from anesthetized mice and were then preserved in liquid nitrogen for further phenotypic analyses. Sham controls were subjected to the same operation procedure but without clamping. For antibody treatment, approximately 1 h before hepatic I/R, mice were administered with isotype antibody (A110-1, BD Biosciences, San Jose city, CA, USA) (800 ug) or P-selectin (RB40.34, Thermo Scientific, Waltham, MA, USA) (200 ug), E-selectin (BE0294, BioXcell, Lebanon, NH, USA) (200 ug), and αvβ3 integrin antibody (RMV-7/2C9.G2, Biolegend, San Diego, CA, USA) (200/200 ug) via intraperitoneal injection(s).
3
Quantifying Regulatory T Cells by Flow Cytometry
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Flow cytometry was used to verify the cell activity and purity after magnetic beads separation. The separated cells were resuspended with DMSO solution and divided into isotype pair tube, CD4+CD25+ tube and sample tube. Added 1 μL FVS510 (BD biosciences, 564406) in three tubes. Added 2 μL PE-labelled CD4 Isotype (BD biosciences, clone A95-1, rat. # 553989) and 5 μL APC-labelled CD25 isotype (BD biosciences, clone A110-1, rat. # 550884) in isotype pair tube. Added 2 μL PE-labelled CD4 (BD biosciences, clone GK1.5, rat. # 561829) and 5 μL APC-labelled CD25 (BD biosciences, clone PC61, rat. # 561048) in CD4+CD25+ tube and sample tube. 1 mL of freshly prepared 1× TF Fix/Perm working buffer was added to each tube, which was mixed and incubated at 2–8 °C in dark for 30 min. Added 1 mL 1× Perm/Wash working buffer into each tube. Added 2 μL BV421-labeled Foxp3 antibody (BD biosciences, clone MF23, rat. # 562996) and 2 μL BV421-labeled Foxp3 isotype antibody (BD biosciences, clone R35-38, rat. # 562603) in sample tube and isotype pair tube, respectively. The solution was stained for 30 min avoiding light, and then was centrifugated for 5 min. Added 0.5 mL PBS into each tube for mixing.
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