Human neutrophils (1 × 107/mL) were loaded with 20 μM 2,7-dichlorofluorescein diacetate (DCFH-DA, Sigma Aldrich) in Hank’s balanced salt solution (HBSS, Cellgro) without Ca2+ and Mg2+ and incubated with rotation at 37°C for 20 min. Neutrophils were washed once with PBS and resuspended in HBSS with Ca2+ and Mg2+ to a density of 1 × 106 cells/well in a white wall 96 well palate (Costar, Princeton, NJ, United States). Multiplicity of infection (MOI) = 1 bacteria per neutrophil was added to each well and was incubated for 30 min at 37°C with 5% CO2. Fluorescence intensity at 485 nm excitation/520 nm emission quantified on an Enspire plate reader (Perkin Elmer).
Hanks balanced salt solution (hbss)
Manufactured by Corning
Sourced in United States
HBSS is a balanced salt solution used in cell culture applications. It provides a controlled ionic environment to maintain the pH, osmolarity, and other key parameters necessary for the growth and maintenance of cells in vitro.
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Lab products found in correlation
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (14 mentions)
Dnase 1, by Roche (13 mentions)
Collagenase d, by Roche (12 mentions)
Hanks balanced salt solution (hbss), by Merck Group (7 mentions)
Hepes, by Corning (7 mentions)
Matrigel, by Corning (6 mentions)
Dulbecco modified eagle medium (dmem), by Corning (6 mentions)
Hanks balanced salt solution (hbss), by Worthington (5 mentions)
Percoll, by GE Healthcare (5 mentions)
Dnase 1, by MP Biomedicals (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (4 mentions)
Collagenase p, by Roche (4 mentions)
Rpmi 1640, by Corning (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Histopaque, by Merck Group (4 mentions)
Collagenase 4, by Merck Group (4 mentions)
Alamar blue, by Thermo Fisher Scientific (4 mentions)
Alamarblue, by Corning (4 mentions)
Cell dissociation buffer, by Merck Group (3 mentions)
192 protocols using hanks balanced salt solution (hbss)
1
Neutrophil Oxidative Burst Assay
2
Isolation and Culture of Intestinal Crypts
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Crypts from the jejunum were isolated immediately after sacrifice as in [19 (link)] with a few modifications. Jejunum was isolated, rinsed with 10mL HBSS (Corning Cellgro, cat# 21-022-CV) cut open longitudinally and gently agitated in 10mL cold HBSS to remove remaining contents. The jejunum was then cut into 1cm pieces and transferred to 3mM EDTA (Sigma, cat# E5134-250G) in HBSS on ice for 30 min with agitation. Tissue pieces were transferred to HBSS and shaken for 3 min. The supernatant was passed through a 70μm filter and intact crypts counted. An appropriate number of crypts were centrifuged at 150g for 5 min. Crypts were resuspended in basement membrane matrix growth-factor reduced, phenol red free Matrigel (Corning, cat# 356231) and 10μL plated into 24 or 48 well dishes. Media (DMEM-F12 with glutamine (ATCC, 30–2006)) contained: B-27 (50X Life Technologies cat# 17504044), N-2 (100X Life Technologies cat# 17502–048) penicillin/streptomycin (100X Gibco, cat#15140–122), HEPES (100mM, Amresco, cat# J848-100ML), gentamicin/amphotericin (500X Life Technologies, cat# J0-0640), EGF (50ng/mL, Life Technologies, cat# PMG8045), Noggin (100ng/mL, Peprotech, cat# 250–38), human R-spondin1 (500ng/mL, Peprotech, cat# 120–38), Y27632 (10μM, Tocris: R&D Systems cat# 1254) and Chir99021 (2.5μM, Stemgent, cat# 04-0004-02).
3
Isolation and Dissociation of Pancreatic Islets
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Islets were isolated as previously described (83 (link)–86 (link)). Following euthanasia, the pancreas was inflated via the common bile duct with ~3 ml of 0.8mg/ml Collagenase P (Roche) or 2.5 ml CIzyme RI (Vitacyte) and 10µg/ml Dnase I (Roche) in HBSS (Cellgro). The pancreas was removed and digested at 37°C. Islets were obtained by density centrifugation and hand-picked under a dissecting microscope. Single cell suspensions were made by digestion using 0.4 Wunsch Units/ml Collagenase D (Roche) and 250 µg/ml Dnase I (Roche) in HBSS (Cellgro) with 10% FBS (Hyclone) at 37°C for 30 min. Islets were dissociated in Cell Dissociation Buffer (Sigma) at 37°C for an additional 30 min.
4
Isolation and Characterization of Murine Islets
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Islets were isolated as previously described (Melli et al., 2009 (link); Friedman et al., 2014 (link)). Briefly, mice were anesthetized with ketamine/xylazine before cervical dislocation. The pancreas was inflated via the common bile duct by adding ∼3 ml of 0.8 mg/ml Collagenase P (Roche) and 10 µg/ml DNase I (Roche) or CIzyme RI (Vitacyte) in HBSS (Cellgro). Following inflation, the pancreas was removed and incubated at 37°C for 10–11 min for Collagenase P digestion or 17 min for CIzyme RI digestion, and islets were isolated by density centrifugation. Islets were handpicked under a dissecting microscope. For experiments in which islets were analyzed using flow cytometry, single-cell suspensions were generated by digestion with 0.4 Wunsch units/ml Collagenase D (Roche) and 250 µg/ml DNase I (Roche) in HBSS (Cellgro) with 10% FBS (Hyclone) at 37°C for 30 min. Following initial digestion, islets were then incubated in Cell Dissociation Buffer (Sigma-Aldrich) at 37°C for an additional 30 min.
5
Visualizing SLC46A2 Localization in HEK293 Cells
6
Enrichment and Implantation of Cells
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Fifty microliters of the RosetteSep cocktail (StemCell Technologies) was added for each 1 ml of blood or DLA product and incubated 20 min at RT. After incubation, the sample was diluted with an equal volume of Hank’s Balanced Saline Solution (HBSS) (Life Technologies, Carlsbad, CA, USA) supplemented with 2% fetal bovine serum (FBS) (Life Technologies). The solution was then carefully layered on top of 15 ml Ficoll-Paque Plus (GE-Healthcare, Little Chalfont, UK) and centrifuged for 20 min at 1200 × g without brake. Enriched cells were collected, washed with 50 ml HBSS/2% FBS, and centrifuged for 5 min at 250 × g. Cells were resuspended in 100 µl of cold HBSS supplemented with 100 µl cold Matrigel (Corning, NY, USA) and kept on ice until implantation in mice.
7
Isolation of Murine Lung Cells
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Before extraction, lungs were perfused with 30 ml normal saline through right ventricle of the heart under 2% isoflurane inhalation anesthesia. Lung tissues were harvested from mice and filled with Ca++ and Mg++ free Hank’s balanced salt solution (HBSS) (Corning; Corning, NY).
They were sliced into small pieces by sterile surgical blade and were digested in HBSS supplemented with 100 U/ml of collagenase I (Worthington Biochemical, Lakewood, NJ) for 30 min at 37 °C with periodic shaking. The resultant fragments were meshed by a 70 µm cell strainer (Corning) and a 10 ml syringe plunger and washed with HBSS. The remaining red blood cells were lysed with lysing buffer (BD Biosciences, San Jose, CA). The numbers of isolated lung cells were counted using a microscope (Eclipse TS100; Nikon, Tokyo, Japan).
They were sliced into small pieces by sterile surgical blade and were digested in HBSS supplemented with 100 U/ml of collagenase I (Worthington Biochemical, Lakewood, NJ) for 30 min at 37 °C with periodic shaking. The resultant fragments were meshed by a 70 µm cell strainer (Corning) and a 10 ml syringe plunger and washed with HBSS. The remaining red blood cells were lysed with lysing buffer (BD Biosciences, San Jose, CA). The numbers of isolated lung cells were counted using a microscope (Eclipse TS100; Nikon, Tokyo, Japan).
8
Mitochondrial Superoxide Evaluation
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500,000 cells were stained with 5 μM MitoSOX Red mitochondrial superoxide indicator (Invitrogen, M36008) in 1 ml HBSS (Corning, 21–023-CV) for 30 minutes at 37⁰C. Following incubation, cells were washed with HBSS, and florescence was measured using flow cytometry as per manufacture’s protocol. Unstained cells were used as a negative control while cells treated with 50 μM Antimycin A were used as a positive control for flow cytometry.
9
Assessing Lipid Peroxidation in Jurkat Cells
10
Lung Slice Preparation and Preservation
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PCLS were prepared as described previously[50 (link), 51 (link)]. Following tracheotomy, the mouse lungs were insufflated with 1% low-melting point agarose in HBSS and placed in cold HBSS (Corning Life Sciences, MA, USA). After the agarose had formed a gel, the two largest lobes were separated and then sectioned into 250 μm thick slices using a Precisionary Instruments VF-300 tissue slicer. Lung slices were cultured in 1:1 DMEM/F-12 supplemented with penicillin, streptomycin, kanamycin, and amphotericin B (Invitrogen, MA, USA). Media was changed once an hour for the first 4 hours. As described in Rosner et al.[52 (link)], the PCLS were then slowly frozen at a rate of 1°C/minute in culture media supplemented with 10% DMSO. For the dose-response and stretch experiments, the PCLS were rapidly thawed in a 37°C water bath and cultured in fresh media for at least 2 hours before experiments were performed.
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