The procedures were performed in accordance with a previously described protocol [28 (link)]. After purification and siRNA transfection as described above, primary astrocytes were cultured with deoxygenated DMEM without glucose and FBS (Gibco, CA, USA) in an incubator (Thermo Scientific) with premixed gas (1 % O2, 94 % N2, 5 % CO2) for 5 h. Then, the cells were given normal DMEM (Gibco, CA, USA) containing 10 % FBS and placed in a CO2 incubator (95 % air and 5 % CO2) for 24 h. Cells in the control group were cultured with normal DMEM and 10 % FBS for the same time. After OGD induction, 0.01, 0.1, 1, 10 mM Sino was immediately used to treat astrocytes for 24 h.
Normal dmem
Manufactured by Thermo Fisher Scientific
Sourced in United States
Normal DMEM is a culture medium commonly used for the in vitro growth and maintenance of various cell lines. It provides the necessary nutrients, salts, and buffering agents to support cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Bovogen (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
5 protocols using normal dmem
1
Astrocyte Hypoxia and Sino Treatment
2
Overexpression of lncRNA in Granulosa Cells
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The lncRNA overexpression vector was constructed using the linear sequence of lnc4040 amplified from GCs by PCR. They were then cloned into the pcDNA3.1-EGFP vector in accordance with the manufacturer's protocol using the NheI and KpnI restriction sites. GCs were collected from follicular fluid and cultured to P2 with 10% FBS (Gibco-BRL, Grand Island, NE, USA) added to normal DMEM (Gibco-BRL, Grand Island, USA). miRNA mimics, mimics NC, and lncRNA overexpression vector were transiently transfected into GCs using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). GCs were collected 48 h after transfection, and proteins were extracted for further experiments.
3
Chlamydia trachomatis L2 Infection
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C.C. trachomatis serovar L2 (ATCC VR-902B) was used to infect cells at a multiplicity of infection (MOI) of ~0.5. Cells were infected 2 hr post-transfection in normal DMEM (Gibco) supplemented with 10% (v/v) FCS (Bovogen) and 2 mM L-glutamine (Invitrogen) in a humidified air/atmosphere (5% CO2) incubator at 37°C. After 2 hr media was replaced with fresh media.
4
Cell Culture and Chemical Treatments
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HEK293T (human embryonic kidney), HeLa (human adenocarcinoma), U87MG (human glioblastoma), and Neuro-2a (mouse neuroblastoma) cells were cultured in normal DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/mL penicillin (Invitrogen) and 100 μg/mL streptomycin (Invitrogen). All cells were cultured an incubator at 37 °C and 5% CO2. Cell treatments are carried out by adding chemicals to the culture media to the levels specified in legends. BHB treatment takes 12 h.
5
Astrocyte Culture and Oxygen-Glucose Deprivation
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Primary astrocytes were removed from the C57L/6J mice cerebellum and cultured using Dulbecco Modified Eagle's Medium (DMEM; Gibco, USA), enriched with Fetal bovine serum (FBS, 10%; Gibco, USA), 5% CO 2 at 37 °C. TNF-α, IL-1β, and -6 levels were estimated with ELISA kits (Sangon Biotech, China). In accordance with the instructions, cells were transfected with Lipofectamine 3000 (Invitrogen, USA) with shRNA-NC, circRIMS, and miR-96-5p inhibitors.
Oxygen glucose deprivation-reperfusion (OGD/RX) was performed as described (32) (link). Briefly, in the absence of glucose and FBS, cells culture is performed in DMEM (Gibco, USA) for 48 h with pre-mixed gas (95% N 2 and 5% CO 2 ) in an incubator at 37 °C. Subsequently, cells were exposed to normal DMEM (Gibco, USA), FBS (10%, Gibco, USA), and placed in an incubator containing air (95%) and CO 2 (5%). Simulated group cells were only cultured in normal DMEM (Gibco, USA) and FBS (fetal bovine serum, 10%; Gibco, USA).
Oxygen glucose deprivation-reperfusion (OGD/RX) was performed as described (32) (link). Briefly, in the absence of glucose and FBS, cells culture is performed in DMEM (Gibco, USA) for 48 h with pre-mixed gas (95% N 2 and 5% CO 2 ) in an incubator at 37 °C. Subsequently, cells were exposed to normal DMEM (Gibco, USA), FBS (10%, Gibco, USA), and placed in an incubator containing air (95%) and CO 2 (5%). Simulated group cells were only cultured in normal DMEM (Gibco, USA) and FBS (fetal bovine serum, 10%; Gibco, USA).
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