Primary mouse hepatocytes were isolated by liberase perfusion as previously described [35 ]. Briefly, the liver of anesthetized mice was first perfused with calcium-free Hanks balanced salt solution supplemented with HEPES and EDTA (HBSS, Biological Industries, Beit HaEmek, Cat# 02–018-1A HEPES Cat# 03–025-1B), followed by perfused liberase digestion (25 μg/mL liberase in HBSS; Biological Industries, Beit HaEmek, Cat# 02–015-1A with 25 mM HEPES). After digestion, the hepatocytes were released by dissociation from the lobes (centrifuged at 50 ×g for 2 min at 4 °C). The pellet from the first centrifugation of hepatocytes was loaded on a 90% Percoll gradient (Sigma-Aldrich; Cat# GE17–0891-01) and centrifuged at 200 ×g for 10 min at 4 °C. The cells were then cultured on 6-well plates at a density of 4 × 105 cells/well with planting medium (DMEM, Biological Industries, Beit HaEmek, Cat# 01–050-1A) supplemented with 100 U/mL penicillin-streptomycin and 5% FBS, which, was changed after 3 h to William's E medium (Rhenium; Cat# 22551022) containing 2 mM l -glutamine and 100 U/mL penicillin-streptomycin. Cells were incubated overnight at 37 °C in a humidified 5%CO2/95% air atmosphere.
Hanks balanced salt solution (hbss)
Manufactured by Sartorius
Sourced in Israel
HBSS is a balanced salt solution used in cell culture and biomedical applications. It provides a physiologically-balanced environment for the maintenance and growth of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Sartorius (3 mentions)
Dulbecco modified eagle medium (dmem), by Sartorius (3 mentions)
L glutamine, by Sartorius (2 mentions)
Complete rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Sartorius (2 mentions)
Mg and ca, by Sartorius (2 mentions)
F200 microplate luminescence reader, by Tecan (1 mentions)
White 96 flat bottom wells, by Corning (1 mentions)
Luminol, by Merck Group (1 mentions)
Microwell 96 well plate, by Thermo Fisher Scientific (1 mentions)
Nylon mesh, by BD (1 mentions)
Truestain fcx, by BioLegend (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Collagenase, by Roche (1 mentions)
Dnase 1, by Roche (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Streptomycin, by Sartorius (1 mentions)
Hepes, by Sartorius (1 mentions)
Sodium pyruvate, by Sartorius (1 mentions)
Non essential amino acids (neaa), by Sartorius (1 mentions)
29 protocols using hanks balanced salt solution (hbss)
1
Primary Mouse Hepatocyte Isolation
2
Primary Mouse Hepatocyte Isolation
3
In Vitro Cerebral Ischemia-Reperfusion Injury
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For the in vitro study, the cerebral I/R injury model was set up by OGD/R as described previously (Tang et al., 2016 (link)). N2a neuroblastoma cells were treated as follows: (i) Control, normal cell; (ii) OGD/R group; and (iii) OGD/R + MSC group (Figure 3A ). For OGD/R group, 1 × 105 N2a cells were grown at the six-well culture plates and then placed into a modular incubator chamber (Billups Rothenberg, Inc., Del Mar, CA, United States) with a gas mixture of 5% CO2 and 95% N2. The culture medium was replaced with deoxygenated glucose-free Hanks’ Balanced Salt Solution (Biological Industries) for 4 h. After OGD, the Hanks’ Balanced Salt Solution was removed and the fresh culture medium (DMEM with 10% FBS) was added back for the re-oxygen and re-glucose.
4
Neutrophil ROS Production Assay
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Purified PD-L1pos and PD-L1neg neutrophils were plated (2 × 105) in 180 µL of HBSS (Biological Industries) in white 96 flat-bottom wells (Corning) containing 50 µM luminol (Sigma). ROS production (chemiluminescence) was measured using a Tecan F200 microplate luminescence reader.
5
Intracutaneous Tumor Inoculation in Mice
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U87 cells were inoculated intracutaneously to the right lower lateral side of the back in a concentration of 5 × 106 cells/100 μl of HBSS (Biological Industries, Kibbutz Beit Haemek, Israel), using a 29-gauge needle. The procedure was performed under anesthesia (xylazine 10 mg/kg, ketamine 100 mg/kg, i.p.(or isoflurane (5% isoflurane for initial anesthesia and 3% isoflurane for the DaRT insertion procedure).
6
Measurement of IFN-γ-Induced Calcium Dynamics
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Intracellular calcium was measured as previously described [25 (link)]. Briefly, to measure the effect of IFN-γ on calcium ER stores, SKMEL28 cells were seeded on black, clear bottomed Nunc™ MicroWell™ 96-Well Plates and grown to confluence for one day, followed by treatment with IFN-γ 100ng/mL or PBS (Control) for 48hr. Prior to the measurements, cells were loaded for 30 min at 37°C with 5μM Fluo-3-AM dissolved in Ca2+-containing Hanks’ Balanced Salt Solution (HBSS) without Phenol Red (pH 7.4) (Biological Industries). Then, Fluo-3-AM was removed by rinsing with HBSS once followed by thermo-equilibration at room temperature for 20 min. At the start of the experiment, the HBSS was replaced with a nominally Ca2+-free Hanks’ Balanced Salt Solution (HBSS) Without Calcium and Magnesium no Phenol Red (Biological Industries) containing 1mM EGTA. Recordings were started with baseline measurements of the fluorophore fluorescence excited at 488 nm and recorded at 535 nm. After 10 min, 1μM Ta or DMSO were added by changing media and continue recording for up to 20 min.
7
Murine Subcutaneous Tumor Inoculation
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Mice were inoculated i.d. with 5 × 105 cells, unless otherwise specified, into the low lateral side of the back in 0.05 ml RPMI or Hanks’ balanced salt solution (HBSS) (Biological Industries).
8
Isolation and Analysis of CNS-Mononuclear Cells
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Mice were euthanized by isoflurane inhalation, perfused, and their brains were collected in cold PBS. The isolated brains were minced and digested for 1 h at 37 °C in Hanks' Balanced Salt Solution (HBSS) (02–017-1A, Biological Industries Israel Beit Haemek LTD, Israel) containing 50 μg/ml DNase I and 100 μg/ml collagenase (Roche, Rotkreuz, Switzerland). The resulting cell suspension was passed through a 70-μm Nylon mesh (Falcon, BD Biosciences, Bedford, MA), pelleted, resuspended in 30% Percoll (GE Healthcare Bio-Sciences, NJ) in HBSS and centrifuged at 15,500 rpm for 30 min at 4 °C. After eliminating the myelin debris, the mononuclear cell phase was collected. CNS-mononuclear cells were washed in a FACS buffer (HBSS, 2% FCS, 10 mM EDTA). After blocking non-specific binding of immunoglobulins to Fc receptors (anti-mouse CD16/32, TrueStain FcX TM, BioLegend), the cells were stained for 15 min at 4 °C with specific antibodies, washed, and analyzed. Multicolor FACS analyses were performed with a Cytoflex.
9
Immunostaining of Primary Neurons
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Primary neurons grown on cover slips, at 4 or 14 DIV were gently washed with warm HBSS (Biological Industries, Beit-Haemek, Israel), fixed with 2% PFA for 20 min at room temperature and permeabilized with 0.5% saponin in 1% (w/v) BSA for 30 min, at room temperature. Cover slips were then incubated for 2 h at room temperature with the indicated primary antibodies (Supplementary Table S2 ) in 1% (w/v) BSA, followed by 3 washes in PBS, 5 min each. Slides were then incubated with appropriate secondary ab, washed and mounted in vectashield® mounting medium (Vector-labs, Burlingame, CA USA).
10
Isolation of Murine Pancreatic Islets
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Mice (7-week-old) were killed by cervical dislocation immediately before islet isolation. The pancreata were perfused with 2–3 ml of collagenase XI (0.5 mg/ml; Sigma). Bile ducts were clamped off at the duodenal insertion to allow cannulation and specific perfusion into the pancreas. Perfused pancreata were digested for a batch-specific optimized time and collagenase activity was stopped with ice-cold HBSS (Biological Industries Inc., Beth Haemek, Israel). Islets were collected by handpicking under a dissecting microscope into RPMI 1640 complete medium, (containing 10%; heat-inactivated FBS GIBCO, Carlsbad, CA), 2mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and non-essential amino acids (Biological Industries).
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