Protein uptake and retention (up to 24 hours) was studied in washed platelets, PMA-treated HEL cells and CD34+ cell–derived MKs suspended in buffer using flow cytometry. Platelet suspensions (1 × 106/mL) were incubated with fluorescence conjugated Cy3-albumin (10 μg/mL), Cy3-fibrinogen (50 μg/mL), or Cy3-IgG (20 μg/mL) at 37°C for different times. HEL cells or MK (1.5 × 105) were incubated with fluorescent-conjugated albumin Alexa Fluor 488 (30 μg/mL), fibrinogen Alexa Fluor 647 (10 μg/mL), or IgG-Alexa Fluor 488 (30 μg/mL) at 37°C. Cells were fixed (2% paraformaldehyde) and uptake-retention was evaluated using a BD LSRII flow cytometry (San Jose, CA) and analyzed with FlowJo software, v10.5.3. The LSRII flow cytometer was calibrated using standard beads, SPHERO Ultra Rainbow Fluorescent Particles (Spherotech, Inc) on each experimental day. Where indicated, cells were incubated (15 minutes, RT) before uptake studies with 30 μM Pitstop 2 (Cayman Chemical).4 (link)
Sphero ultra rainbow fluorescent particles
Manufactured by Spherotech
SPHERO Ultra Rainbow Fluorescent Particles are a range of highly uniform, monodisperse fluorescent microspheres. They are available in a variety of colors and sizes. The particles can be used as fluorescent labels, calibration standards, and in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Pitstop 2, by Cayman Chemical (2 mentions)
Control sirna, by Santa Cruz Biotechnology (1 mentions)
Poly l lysine solution, by Merck Group (1 mentions)
Human fibronectin, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Enzo Life Sciences (1 mentions)
Human fibrinogen, by Prolytix (1 mentions)
Pharmingen stain buffer, by BD (1 mentions)
Ghost dye violet 510 fixable live dead stain, by Cytek Biosciences (1 mentions)
3 protocols using sphero ultra rainbow fluorescent particles
1
Protein Uptake Dynamics in Platelets, HEL Cells, and MKs
2
Cell Adhesion and Endocytosis Assay
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The following reagents were obtained: human fibronectin (EMD Millipore, Burlington, MA), poly-L-lysine solution (Sigma Life Science, St. Louis, MO), SPHERO Ultra Rainbow fluorescent particles (Spherotech, Inc, Lake Forest, IL), phorbol 12-myristate 13-acetate (PMA) (Enzo Life Sciences, Farmingdale, NY), human fibrinogen (Haematologic Technologies, Essex Junction, VT), and Pitstop 2 (Cayman Chemical, Ann Arbor, MI). Small interfering RNAs (siRNAs) against RUNX1, CAV1, FLOT1, IFITM3 interferon induced transmembrane protein 3, and CLTC clathrin heavy chain, and control siRNAs, were purchased from Santa Cruz Biotechnology Inc, (Dallas, TX). Supplemental Table 1 presents the antibodies and other reagents used.
3
Dissociation and Staining of Primary Cells
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Native tissue was cut into ~ 1 mm diameter pieces and digested in 50 mg/ml collagenase and 1000 U/ml DNase I for up to 2 h in a 37 °C water bath, mixed vigorously every 10–15 min and monitored closely until the bulk of tissue disintegrated into single cell suspension. The samples were then resuspended with repeated pipetting using a 1000 ul tip followed by processing through a 70 mm cell strainer. PDCOs were spun out of the matrix followed by digestion with Trypsin–EDTA (HyClone, Thermo Fisher Scientific) and resuspension in BD Pharmingen™ Stain Buffer (BD Biosciences, CA).
Cells were stained with Ghost Dye™ Violet 510 fixable live/dead stain (Tonbo Biosciences, Sand Diego, CA) and fluorescently labeled antibodies listed in Online Resource Tables S2 and S3. For intracellular staining, fixation and permeabilization was performed following the manufacturer’s protocol (eBioscience, Thermo Fisher Scientific, MA). Samples were acquired following a pre-acquisition instrument standardization with SPHERO™ Ultra Rainbow Fluorescent Particles (Spherotech, IL) on a BD LSR II instrument at the UWCCC Flow Cytometry Laboratory and data were analyzed by FlowJo v9.6 (BD Biosciences, CA). Gating controls included Internal Negative Controls (INC) and Fluorescent Minus One (FMO) controls.
Cells were stained with Ghost Dye™ Violet 510 fixable live/dead stain (Tonbo Biosciences, Sand Diego, CA) and fluorescently labeled antibodies listed in Online Resource Tables S2 and S3. For intracellular staining, fixation and permeabilization was performed following the manufacturer’s protocol (eBioscience, Thermo Fisher Scientific, MA). Samples were acquired following a pre-acquisition instrument standardization with SPHERO™ Ultra Rainbow Fluorescent Particles (Spherotech, IL) on a BD LSR II instrument at the UWCCC Flow Cytometry Laboratory and data were analyzed by FlowJo v9.6 (BD Biosciences, CA). Gating controls included Internal Negative Controls (INC) and Fluorescent Minus One (FMO) controls.
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