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Proteins from heart tissue and NRCMs were extracted in buffer containing 1% sodium deoxycholate, 10 mmol/L Na[sub]4P[sub]2O[sub]7, 1% Triton X-100, 10% glycerol, 150 mmol/L NaCl, 5 mmol/L EDTA-Na[sub]2, 50 mmol/L Tris (pH 7.4), 0.1% SDS, 50 mmol/L NaF, 1 mmol/L Na[sub]3 VO[sub]4, 1 mmol/L PMSF, and protease inhibitor cocktail (Roche, Switzerland).
NRCMs were harvested at the indicated time points, washed, fixed, and centrifuged (1500 x g ), and then labeled with propidium iodide (Invitrogen).
NRCMs were seeded in 24-well plates at a concentration of 1 x 105 cells/ml.3 H-leu (1 uci/ml) was added for 12 h before the cells were harvested.
qRT-PCR was performed to detect the expression of the screened targets following overexpression of miR-24 in NRCMs. The results showed that the expression of CDKN1B (p27, Kip1) was significantly decreased (0.69 [+ or -] 0.07 vs.
Therefore, the optimum concentration of mice monoclonal [[beta].sub.1]-AA leading to the NRCMs beating frequency was 0.1 [micro]m (detected from 1 nM to 1 [micro]M).
The picture in the supplementary material reflects the increased beating frequency of NRCMs stimulated with different concentrations of [[beta].sub.1]-AA.
The video file in the supplementary material mainly indicates that we successfully extracted NRCMs from the experiments and measured the beating frequency.
Caption: Figure 2: Relationship between different reactant concentrations and beating frequency of NRCMs. (a) Control model in three minutes.
Briefly, NRCMs were collected and incubated at 37[degrees]C for 30 min with JC-1 (1x), washed twice with PBS for detection of the fluorescent ratio by flow cytometry.
The mitochondria and NRCMs with under hypoxic and ischemic conditions treated with different concentrations of GS-Rb1 for 24 h were harvested and lysed with Cell Lysis Buffer for Western blotting and IP.
Briefly, NRCMs lysates were prepared after treatment with different dose of GS-Rb1 for 24 h under hypoxia/ischemia conditions.
Effect of GS-Rb1 on the Viability of NRCMs. The effect of GS-Rb1 on NRCMs viability was examined by the MTT assay (Figure 1(a)).
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