RIPA buffer added to HT22 cells (Invitrogen; USA) to collect the total protein; then the protein concentrations were determined assessed using the BCA method (Invitrogen, USA). The protein samples were mixed with a 5x loading buffer and denatured at the boil. The proteins were separated by 15% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Next, the membranes were incubated with primary antibodies (Bcl-2, Bax, caspase-3, BDNF, NGF, SIRT1, Wnt1, β-catenin, Shh, Patch, and GAPDH) at 4°C overnight. Then, the next day, the membranes were washed with PBS and incubated with secondary antibodies for 1 h. Finally, the protein bands were measured using the ImageJ software. The data were collected from at least three independent experiments.
Ht22 cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HT22 cells are an immortalized mouse hippocampal neuronal cell line. They are commonly used in cell-based assays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca method, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
U0126, by Selleck Chemicals (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Ht22 cells, by Procell (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
4 protocols using ht22 cells
1
Western Blot Analysis of Apoptosis and Neurogenesis
2
Lipid Droplet Accumulation in HT22 Cells
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HT22 cells (Procell, Wuhan, China, Cat# CL-0697, RRID: CVCL_HT22, identity confirmed by the short tandem repeat method) were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA, Cat# D0819) with 10% fetal bovine serum (Invitrogen, Waltham, MA, USA, Cat# 10100147) and 100 U/mL penicillin-streptomycin (Invitrogen, Cat# 15070063) at 37°C in 5% CO2.
To induce LD accumulation, HT22 cells in the MD group were treated with 500 μg/mL of MD for 24 hours. HT22 cells in the MD + BRO group were treated with 500 μg/mL of MD and 500 nM of BRO for 24 hours. For Control group, the growth medium was changed, but no treatment was applied.
To inhibit phosphorylation, HT22 cells were cultured in growth medium containing 10 μM U0126 (Selleck, Huston, TX, USA, Cat# S1102) and 500 μg/mL of MD for 24 hours.
To induce LD accumulation, HT22 cells in the MD group were treated with 500 μg/mL of MD for 24 hours. HT22 cells in the MD + BRO group were treated with 500 μg/mL of MD and 500 nM of BRO for 24 hours. For Control group, the growth medium was changed, but no treatment was applied.
To inhibit phosphorylation, HT22 cells were cultured in growth medium containing 10 μM U0126 (Selleck, Huston, TX, USA, Cat# S1102) and 500 μg/mL of MD for 24 hours.
3
Primary Neuron Culture and Cell Lines
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Primary hippocampal neurons were cultured as previously described (Zhang et al., 2014 ). HT‐22 cells were purchased from Life Technologies, and human embryonic kidney 293A cells were purchased from American Type Culture Collection (ATCC).
4
HT-22 Cell Culture Protocol
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HT-22 cells were obtained from Life Technologies (Waltham, MA, USA) and cultured in complete Dulbecco's Modification of Eagle's Medium (DMEM, Gibco, Invitrogen GmbH, Karlsruhe, Germany) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin at 37°C, 5% CO 2 , and 95% saturated humidity. When the cells reach the logarithmic growth phase, they are subcultured in proportion.
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