Hippocampal cultures were made using previously established methods (31 ). Briefly, brains from P0-P1 C57Bl/6J pups (The Jackson Laboratory, Bar Harbor, ME) were isolated, and the hippocampus was dissected in ice-cold HBSS solution (Sigma, St. Louis, MO) supplemented with 20% FBS and NaHCO3 (4.2mM), HEPES (1mM; Sigma), pH 7.4. Dissected hippocampi were digested for 10 min with 0.25% Trypsin (Thermo Fisher Scientific), then washed and dissociated using fire polished Pasteur pipettes of decreasing diameter in ice cold HBSS containing DNase (1500 U; Sigma). The cells were pelleted, resuspended in plating media and plated at a density of 4–5×105 cells/12-mm coverslip (Electron Microscopy Sciences, Hatfield, PA) coated with poly-Ornithine (0.1mg/ml; Sigma; Cat. #4638) and laminin (5μg/ml; Thermo Fisher Scientific). Cells were allowed to adhere for 15 min before addition of 0.5ml of plating media containing Neurobasal supplemented with 1X B27, 2mM Glutamax, 0.5mg/ml Pen/Strep and 5% FBS (all from Thermo Fisher Scientific) for the first 24 h. Half of the media was removed and replaced with serum-free media after 24 h. Half of the media was removed and replaced after 48 h supplemented with 4μM cytosine 1-β-d-arabinofuranoside (Ara-C; Sigma). Neurons were fed by replacing half the volume of spent media with fresh media without serum or Ara-C every week thereafter.
Hbss solution
Manufactured by Merck Group
Sourced in United States
HBSS (Hank's Balanced Salt Solution) is a widely-used cell culture medium that provides a balanced ionic and nutrient environment for the maintenance and growth of cells in vitro. It is a sterile, isotonic solution containing inorganic salts, glucose, and phenol red as a pH indicator. HBSS is designed to maintain the physiological pH, osmolarity, and ionic balance necessary for the survival and normal function of cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Pen strep, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Laminin, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
P0 p1 c57bl 6j pups, by Jackson ImmunoResearch (2 mentions)
12 mm coverslip, by Electron Microscopy Sciences (2 mentions)
Cytosine 1 β d arabinofuranoside ara c, by Merck Group (2 mentions)
Neurobasal, by Thermo Fisher Scientific (2 mentions)
Poly ornithine, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
Nahco3, by Merck Group (2 mentions)
Dnase, by Merck Group (2 mentions)
Human tnf α elisa kit, by ABclonal (1 mentions)
Mouse tnf αelisa kit, by ABclonal (1 mentions)
Valinomycin, by Merck Group (1 mentions)
Apo brdu tunel assay kit, by Thermo Fisher Scientific (1 mentions)
Pentabromophenol, by LGC (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Caspase 3 fluorimetric assay kit, by Merck Group (1 mentions)
5 protocols using hbss solution
1
Murine Hippocampal Primary Culture Protocol
2
TNF-α Quantification in Cell Supernatant and Placenta
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The cell supernatant was collected and centrifuged at 1000× g for 10 min at 4°C to remove particulates. The concentration of TNF-α in the cell supernatant was determined by human TNF-α ELISA kit (Abclonal Technology, RK00030) according to the manufacturer’s instructions. For mouse placenta tissue, the placentas were cut into pieces (~1 mm3) and homogenized in pre-cooling HBSS solution (Sigma) on ice. The tissue suspension was then sonicated with an ultrasonic cell disrupter till the solution is clarified, and centrifuged at 10,000× g for 10 min at 4°C. Collect the supernatant for subsequent experiments. The concentration of TNF-α was determined by mouse TNF-α ELISA kit (Abclonal Technology, RK00027) according to the manufacturer’s instructions.
3
Intracellular ROS Production Assay
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Intracellular ROS production was measured by a CM-H2DCFDA-based General Oxidative Stress Indicator assay (Invitrogen). For this purpose, HCPEpiCs (10,000 per well) were sub-cultured into PLL-coated 96-well plates (SPL Life Sciences) and confluent cells were exposed to IVH-III, IVH-IV, or non-IVH control CSF samples (10 v/v %) for 1 h and 4 h. After treatment, cells were washed with HBSS solution (Sigma-Aldrich) two times and then loaded with CM-H2DCFDA (10 µmol/L) for 30 min at 37 °C in the dark. The fluorescence intensity was detected every 30 min for 3 h by applying 488-nm excitation and 533-nm emission wavelengths using a Beckman Coulter DTX 880 analyzer (Beckman Coulter, Pasadena, CA, USA).
4
Apoptosis Induction by Brominated Phenols
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2,4,6-Tribromophenol (2,4,6-TBP) and pentabromophenol (PBP) of 99% purity were purchased from LGC Standards (Teddington, UK). HBSS solution, valinomycin, pluronic F-127, propidium iodide, Hoechst 33342, and a Caspase-3 Fluorimetric Assay Kit were bought from Sigma-Aldrich (St. Louis, MO, USA). FITC Annexin V Apoptosis Detection Kit and Perm/Wash Buffer were bought from Becton Dickinson (Franklin Lakes, NJ, USA). MitoLite Red CMXRos was obtained from AAT Bioquest (Sunnyvale, CA, USA). Fluo-3/AM was obtained from PromoCell (Heidelberg, Germany). A Caspase-8 Fluorimetric Assay Kit, as well as a caspase-9 chromogenic substrate and caspase-9 inhibitor were purchased from BioVision (San Francisco, CA, USA). An APO-BrdU TUNEL Assay Kit and PARP-1 (cleaved Asp214) monoclonal antibody (HLNC4) were obtained from Thermo-Fisher (Waltham, MA, USA). Lymphocyte separation medium (LSM) (1.077 g/cm3) and RPMI medium with L-glutamine were purchased from Cytogen (Seoul, South Korea). Camptothecin was obtained from Pol-Aura, Poland, while ionomycin was purchased from Biokom (Janki, Poland). Other chemicals were of analytical grade and were obtained from POCH, Poland, and Roth, Germany.
5
Hippocampal Neuron Culture from Neonatal Mice
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Hippocampal cultures were made using previously established methods (Chander et al., 2019) . Briefly, brains from P0-P1 C57Bl/6J pups (The Jackson Laboratory, Bar Harbor, ME) were isolated, and the hippocampus was dissected in ice-cold HBSS solution (Sigma, St. Louis, MO) supplemented with 20% FBS and NaHCO3 (4.2mM), HEPES (1mM; Sigma), pH 7.4. Dissected hippocampi were digested for 10 min with 0.25% Trypsin (Thermo Fisher Scientific), then washed and dissociated using fire polished Pasteur pipettes of decreasing diameter in ice cold HBSS containing DNase (1500 U; Sigma). The cells were pelleted, resuspended in plating media and plated at a density of 4-5×105 cells/12-mm coverslip (Electron Microscopy Sciences, Hatfield, PA) coated with poly-Ornithine (0.1mg/ml; Sigma; Cat. #4638) and laminin (5μg/ml; Thermo Fisher Scientific). Cells were allowed to adhere for 15 min before addition of 0.5ml of plating media containing Neurobasal supplemented with 1X B27, 2mM Glutamax, 0.5mg/ml Pen/Strep and 5% FBS (all from Thermo Fisher Scientific) for the first 24h. Half of the media was removed and replaced with serum-free media after 24h. Half of the media was removed and replaced after 48h supplemented and with 4µM cytosine 1-β-d-arabinofuranoside (Ara-C; Sigma). Neurons were fed by replacing half the volume of spent media with fresh media without serum or Ara-C every week thereafter.
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