PPD was synthesized from expired clinical samples of doxorubicin (FeRx Inc., Aurora, CO) as previously described.27 (link) Fulvestrant was purchased from Selleckchem. Poly(d ,l -lactide-co-2-methyl-2-carboxy-trimethylene carbonate)-graft-poly(ethylene glycol) (PLAC–PEG) was synthesized by ring-opening polymerization using a pyrenebutanol initiator to a molecular weight of 12 000 g/mol and conjugated with an average of three PEG chains/backbone (10 000 g/mol PEG) as previously described.28 Polysorbate 80 (H2X, UP80) was purchased from NOF America Corporation. Vitamin E-PEG 1000 (VitEPEG), Pluronic F68, Pluronic F127, Brij L23, and Brij 58 were purchased from Sigma-Aldrich. McCoy’s 5A cell culture media, CholEsteryl BODIPY 542/563 C11, and Hoechst 33342 were purchased from Thermo Fisher Scientific. The SKOV-3 cell line was purchased from ATCC. Charcoal stripped fetal bovine serum and Hank’s balanced salt solution were purchased from Wisent Bioproducts.
Hank s balanced salt solution
Manufactured by Wisent
Sourced in Canada, Cameroon
Hank's Balanced Salt Solution is a widely used cell culture media component. It provides a balanced salt composition to maintain the osmotic pressure and pH of cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Pluronic f 127, by Merck Group (1 mentions)
Skov3 cell line, by American Type Culture Collection (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Doxorubicin, by Selleck Chemicals (1 mentions)
Brij l23, by Merck Group (1 mentions)
Cholesteryl bodipy 542 563 c11, by Thermo Fisher Scientific (1 mentions)
Fulvestrant, by Selleck Chemicals (1 mentions)
Charcoal stripped fetal bovine serum, by Wisent (1 mentions)
Brij 58, by Merck Group (1 mentions)
Alexa fluor 488, by Merck Group (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Coolsnap hq digital camera, by Olympus (1 mentions)
Macs ls column, by Miltenyi Biotec (1 mentions)
Macs monocyte isolation kit 2, by Miltenyi Biotec (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Spinning disk confocal microscope, by Quorum Technologies (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
4 protocols using hank s balanced salt solution
1
Nanoparticle Delivery of Doxorubicin Derivative
2
Imaging B2 Receptor-Mediated Endocytosis
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B2 receptor-mediated transport of immune complexes constructed at the cell surface was previously reported (Bawolak et al., 2011 (link)). Intact HEK 293a cells stably expressing the N-terminally myc-tagged B2 receptor construction (human sequence, Charest-Morin et al., 2015 (link)) were treated with the monoclonal anti-myc antibody (clone 4A6) conjugated with AlexaFluor-488 (Millipore, dilution 1:500) added to the culture medium. Cells were further incubated at 37°C. Without rinsing, a selected antagonist or their DMSO vehicle was added 5 min later, and BK (100 nM) or its saline vehicle 15 min later. After an additional 30 min incubation period at 37°C, cells were rinsed 3 times with Hank's balanced salt solution (Multicell Wisent, St. Bruno, Canada) and photographed in transmission and epifluorescence using an Olympus BX51 microscope coupled to a CoolSnap HQ digital camera (filters for AlexaFluor 488: excitation 460–500 nm, emission 510–560 nm).
3
Isolation and Stimulation of Human Blood Monocytes
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Total blood mononuclear cells were isolated from the blood of healthy donors using lymphocyte separation Medium 1077 (Sigma) and washed twice in Hank's balanced salt solution (Wisent). Cells were cultured in RPMI 1640 medium, 20% heat-inactivated FBS, and 10% heat-inactivated human serum for 2 h before use. Monocytes were washed from nonadherent cells with phosphate-buffered saline (PBS). Monocytes were then enriched from peripheral blood mononuclear cells using the MACS Monocyte Isolation Kit II and MACS LS Columns (Miltenyi Biotec, Auburn, CA, USA), yielding an average purity of 98%. The purity was assessed by flow cytometric analyses as recommend by the manufacturer, and isolated monocytes were fluorescently stained with CD14-FITC and anti-Biotin-PE that labeled nonmonocytes. Blood monocytes were stimulated for the indicated times using 1 μM PGE2 and/or 1 μM T0901317.
4
Spinning-Disk Confocal Microscopy Protocol
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For imaging, cells grown on glass coverslips were mounted in a Chamlide magnetic chamber (Seoul, South Korea), overlaid with 1 ml of Hanks’ balanced salt solution (Wisent Bioproducts) and maintained at 37ºC. Fluorescence microscopy was performed using spinning-disk confocal microscopes (Quorum Technologies, Guelph, Canada). Our systems are based on an Axiovert 200M microscope (Carl Zeiss) equipped with a 63× oil-immersion objective (numerical aperture 1.4) and a 1.5× magnifying lens. The microscopes carry a motorized XY stage (Applied Scientific Instrumentation, Eugene, OR), a Piezo Z-focus drive, and diode-pumped solid-state lasers emitting at 440, 491, 561, 638, and 655 nm (Spectral Applied Research, Richmond Hill, Canada). Images were recorded with back-thinned, cooled charge-coupled device cameras (Hamamatsu Photonics) under command of the Volocity software (version 6.2.1; PerkinElmer, Woodbridge, Canada). Selection of regions of interest, fluorescence intensity measurements, and brightness/contrast corrections were performed with ImageJ (version 1.48; National Institutes of Health, Bethesda, MD). Brightness and contrast parameters were adjusted across entire images and without altering the linearity of mapped pixel values. All images are representative of at least 15 determinations from three separate experiments.
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