Mycoplasma contamination affects cell characteristics and decreases the sensitivity of BV2 microglia to LPS stimulation

Feng, Nianhua; Huang, Xiaoxi; Jia, Yanjun

doi:10.1007/s10616-019-00311-8

Cited by 11 publications

(8 citation statements)

References 37 publications

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“…Microglia was activated and then secreted several pro-inflammatory cytokines during the occurrence of inflammation 7. In addition, nuclear factor (NF)-κB is a transcription factor, which plays an important role in the inflammatory response of microglia cells 8. NF-κB could regulate multiple inflammatory cytokines including IL-2, IL-6, IL-1β and TNF-α 9,10.…”

Section: Introductionmentioning

confidence: 99%

<p>Dexmedetomidine Attenuates Neuroinflammation In LPS-Stimulated BV2 Microglia Cells Through Upregulation Of miR-340</p>

Yang

Zhu

et al. 2019

DDDT

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BackgroundDexmedetomidine (Dex) was reported to exhibit anti-inflammatory effect in the nervous system. However, the mechanism by which Dex exhibits anti-inflammation effects on LPS-stimulated BV2 microglia cells remains unclear. Thus, this study aimed to investigate the role of Dex in LPS-stimulated BV2 cells.MethodsThe BV2 cells were stimulated by lipopolysaccharides (LPS). BV2 cells were infected with short-hairpin RNAs targeting NF-κB (NF-κB-shRNAs) and NF-κB overexpression lentivirus, respectively. In addition, miR-340 mimics or miR-340 inhibitor was transfected into BV2 cells, respectively. Meanwhile, the dual-luciferase reporter system assay was used to explore the interaction of miR-340 and NF-κB in BV2 cells. CCK-8 was used to detect the viability of BV2 cells. In addition, Western blotting was used to detect the level of NF-κB in LPS-stimulated BV2 cells. The levels of TNF-α, IL-6, IL-1β, IL-2, IL-12, IL-10 and MCP-1 in LPS-stimulated BV2 cells were measured with ELISA.ResultsThe level of miR-340 was significantly upregulated in Dex-treated BV2 cells. Meanwhile, the level of NF-κB was significantly increased in BV2 cells following infection with lenti-NF-κB, which was markedly reversed by Dex. LPS markedly increased the expression of NF-κB and proinflammatory cytokines in BV2 cells, which were reversed in the presence of Dex. Moreover, miR-340 mimics enhanced the anti-inflammatory effects of Dex in LPS-stimulated BV2 cells via inhibiting NF-κB and proinflammatory cytokines. Furthermore, Dex obviously inhibited LPS-induced phagocytosis in BV2 cells.ConclusionTaken together, our results suggested that Dex might exert anti-inflammatory effects in LPS-stimulated BV2 cells via upregulation of miR-340. Therefore, Dex might serve as a potential agent for the treatment of neuroinflammation.

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Section: Introductionmentioning

confidence: 99%

<p>Dexmedetomidine Attenuates Neuroinflammation In LPS-Stimulated BV2 Microglia Cells Through Upregulation Of miR-340</p>

Yang

Zhu

et al. 2019

DDDT

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“…Its small size (0.3-1 μm) and non-rigid cell wall makes it also hard to remove by filtration. Mycoplasma infection has a pleiotropic effect on cellular physiology including altered metabolism, DNA, RNA and protein synthesis and proand anti-inflammatory effects [1][2][3]. U937 human monocytic cells, the cell-type used in this study, respond to the Mycoplasma infection by producing monocyte chemotactic protein-1, matrix metalloproteinase-12 [4] and interleukin-1β [5].…”

Section: Introductionmentioning

confidence: 99%

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

et al. 2019

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Objective: We aim to directly detect Mycoplasma DNA in a U937 suspension cell culture without using DNA purification. In order to make Mycoplasma contamination monitoring easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit. We compared the sensitivity of direct qPCR against qPCR with a purified DNA template. Results: Our findings indicate that qPCR worked optimally with a 6 μl sample volume and a 52 °C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 to 20 s without any major decrease in reaction sensitivity. The total cycle time of optimized direct qPCR was 65 min. The optimized qPCR protocol was used to detect Mycoplasma DNA before and after DNA purification. Our findings indicate that direct qPCR had a higher sensitivity than regular qPCR. Ct levels produced by direct qPCR with 6 μl templates were almost identical to Ct levels produced by regular qPCR with DNA purified from a 60 μl cell culture sample (23.42 vs 23.49 average Ct levels, respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from U937 cell cultures.

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“…pleiotropic effect on cellular physiology including altered metabolism, DNA, RNA and protein synthesis, pro-and anti-inflammatory effects [1][2][3]. U937 human monocytic cells, the cell-type used in this study, respond to the Mycoplasma infection by producing monocyte chemotactic protein-1, matrix metalloproteinase-12 [4] and interleukin-1 [5].…”

Section: Introductionmentioning

confidence: 99%

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

Baaity

Breunig

Önder

et al. 2019

Preprint

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Objective In order to make the Mycoplasma contamination monitoring task easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit to detect Mycoplasma DNA without DNA purification in a U937 suspension cell culture. We compared the sensitivities of the direct qPCR and the qPCR with the purified DNA template. Results Our findings indicate that qPCR worked optimally with a 6 ml sample volume and a 52 o C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 sec to 20 sec without any major decrease in the reaction sensitivity. The total cycle time of the optimized direct qPCR was 65 minutes. The optimized qPCR protocol was used to detect Mycoplasma DNA directly and after DNA purification. Our findings indicate that the direct qPCR had a higher sensitivity compared to the regular qPCR. The Ct levels produced by the direct qPCR with 6 ml templates were almost identical to the Ct levels produced by the regular qPCR with DNA purified from a 60 ml cell culture sample (23.42 vs 23.49 average Ct levels respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from the U937 cell cultures.

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Mycoplasma contamination affects cell characteristics and decreases the sensitivity of BV2 microglia to LPS stimulation

Cited by 11 publications

References 37 publications

<p>Dexmedetomidine Attenuates Neuroinflammation In LPS-Stimulated BV2 Microglia Cells Through Upregulation Of miR-340</p>

<p>Dexmedetomidine Attenuates Neuroinflammation In LPS-Stimulated BV2 Microglia Cells Through Upregulation Of miR-340</p>

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures

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