Neonatal rat primary microglia isolated from mixed glial cultures were seeded onto poly-l -lysine-pretreated 24-well plates at 2 × 105 cells per well and grown in culture medium for microglia (RPMI 1640 medium supplemented with 10% FCS, 1 mM l-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 mM β-mercaptoethanol, 100 U ml−1 penicillin, and 100 mg ml−1 streptomycin). The cells were allowed to attach overnight. A total of 100 nM of miR-210 mimic (Qiagen), negative mimic (Qiagen), LNA-anti-miR-210 (Exiqon), LNA scramble control (Exiqon), On-Target plus rat SIRT1 siRNA (Dharmacon), or control siRNA (Dharmacon) were used for in vitro transfection. Primary microglia transfection was performed with HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. For microglia M1 activation, cells were recovered overnight after 6 h transfection and then stimulated by adding LPS (0.5 ng/ml, Sigma) and IFN-γ (0.1 ng/ml, PeproTech). At the desired time points, microglia were collected for analysis.
Mir 210 mimic
Manufactured by Qiagen
Sourced in United States
The MiR-210 mimic is a synthetic RNA molecule designed to mimic the function of the natural miR-210 microRNA. MicroRNAs are small, non-coding RNA molecules that play a role in regulating gene expression. The MiR-210 mimic is intended for use in research applications to study the functional effects of increased miR-210 expression.
Automatically generated - may contain errors
Lab products found in correlation
Hiperfect transfection reagent, by Qiagen (5 mentions)
Lna scramble control, by Qiagen (3 mentions)
Negative mimic, by Qiagen (2 mentions)
Control sirna, by Horizon Discovery (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Mir 210 lna, by Qiagen (2 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Lna anti mir 210, by Qiagen (1 mentions)
Lipopolysaccharides (lps), by Qiagen (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Mirna target ip kit, by Active Motif (1 mentions)
Phenol chloroform isoamyl alcohol, by Thermo Fisher Scientific (1 mentions)
Complete lysis buffer, by Active Motif (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Sc 28860, by Santa Cruz Biotechnology (1 mentions)
Mitochondrial ros detection assay kit, by Cayman Chemical (1 mentions)
6 protocols using mir 210 mimic
1
Microglia Activation Modulation Protocol
2
Investigating Microglial Response to LPS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BV2 mouse microglia cell line was provided by Dr. Grace Y. Sun (University of Missouri). BV2 cells retain many morphological and functional properties of primary microglia [17 (link)]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone Co.) with 10 % fetal bovine serum (FBS; Hyclone Co.), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma) in a humidified incubator at 37 °C with 5% CO2. To determine the effect of liposaccharide (LPS, Sigma) concentration on microglia stimulation, BV2 cells with 80–90% confluency were incubated with multiple doses (0, 10, 100, or 500 ng/ml) of LPS for 6 h. A total of 100 nM of miR-210 mimic, negative mimic (Qiagen), miR-210-LNA, LNA scramble control (Exiqon), TET2 siRNAs (si. Tet2), or control siRNA (si. Ctrl, Dharmacon) were used for in vitro transfection. Transfection was performed with HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. BV2 cells were starved in serum-free medium for 2 h before transfection. At 24 h after transfection, cells were stimulated with or without LPS (1 or 500 ng/ml) for 6 h and then collected for assays.
3
Modulating miR-210 in Primary Neurons
4
Rat Microglia miR-210 Regulation of SIRT1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immortalized rat microglia (Applied Biological Materials) were seeded onto collagen I-pretreated 6-well plates (Thermo Fisher) at 6 × 105 cells per well and transfected with 100 nM of either miR-210 mimic (Qiagen) or negative mimic (Qiagen) by using HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. Cells were harvested 24 h after transfection and washed in ice-cold PBS followed by complete lysis buffer (Active Motif) at 4 °C for 10 min. RISC-IP of the lysate was conducted using the miRNA Target IP Kit (Active Motif) following the manufacturers’ instructions. The RNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1, Fisher Scientific) once, chloroform once and precipitated and resuspended in RNase-free water. The precipitated RNA was subjected to RT-qPCR using primers specific for the rat SIRT1 3′UTR. β-actin was used as an internal control. The relative abundance of SIRT1 transcript pulled down by Ago1/2/3 antibody was calculated by the 2−ΔΔCT method and is presented as the fold induction relative to the control. Primers are presented in Supplementary Table 1 .
5
Transfection of miR-210 in Cells
6
Mitochondrial ROS Detection Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Euthasol® (pentobarbital sodium and phenytoin sodium) was obtained from (Virbac Corporation, Westlake, TX, USA). The mitochondrial ROS Detection Assay Kit and mitoquinone mesylate (MitoQ) were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Oligomycin, carbonyl cyanide‐4(trifluoromethoxy) phenylhydrazone (FCCP) and rotenone/antimycin A were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), Fluo‐4 AM, phenol, chloroform and isoamyl alcohol were obtained from Fisher Scientific (Waltham, MA, USA). Antibodies sc‐28860 and sc‐373694 were obtained from Santa Cruz (Dallas, TX, USA). miR‐210 mimic, AllStarsNegative Control siRNA, and HiPerfect transfection reagent were obtained from Qiagen (Germantown, MD, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!