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MessagePosté le: Mar 21 Oct - 07:41 (2014) Sujet du message: Briefly, 1脳105 HEY cells or ascites derived tumor cells unt Répondre en citant

 Around the following day right after plating, cells had been sub jected to unique treatment options. Treatment method of the AP24534 FGFR 阻害剤 BV 2 microglial cell culture The dose and time dependent results of LPS on ROS manufacturing in BV two cells have been carried out to deter mine the optimum helpful concentration and publicity time of LPS. BV 2 cells were incubated in medium con taining LPS at a variety of concentrations for 6 h. Subsequently, BV two cells have been handled with LPS at one ugml for two, four, 6, 12 and 24 h to establish the time dependent results of LPS. In an effort to study the impact of dexamethasone within the ROS manufacturing in LPS activated BV 2 cells, the cells have been washed with 0. one M phosphate buffered saline, followed by incubation in medium containing one ugml LPS for six h, with or with no thirty min pre incubation of Dex at numerous concentrations.

To ascertain the involvement of MAPK pathway in ROS manufacturing and iNOS mRNA expression, BV two cells had been incubated with both SP600125 or SB203580, inhibitors of JNK and p38 respectively, for 30 min just before LPS treatment method for 6 h. Measurement of ROS production in BV 2 microglia by Flow cytometry Intracellular ROS production was determined by detect ing the AT-406 ic50 fluorescent intensity of two, seven dichlorofluorescein, the oxidized product or service of your fluoroprobe 5 chloromethyl two, seven dichlorodihydrofluorescein diace tate. Briefly, BV 2 cells grown in one hundred mm2 culture dish on the density of 1. 0106 have been treated as described over.

Subsequently, the cells had been trypsinized into dis sociated cells and collected by centrifugation right into a 15 ml Falcon tube. The dissociated cells had Akt1 阻害剤 been re suspended in 0. 5 ml of pre warmed PBS containing CM H2DCFDA and allowed to recover for 40 min while in the darkness. Immediately after the incubation, the cells have been immedi ately measured having a flow cytometer with excitation at 488 nm and emission at 535 nm as well as information obtained was analyzed with Summit V4. 2 computer software. The amount of ROS production was deemed to become straight proportional on the fluores cence intensity. RNA isolation and Actual time RT PCR Complete RNA from BV2 cells was extracted with RNeasy Mini Kit in accordance towards the producers guidelines and quantified spec trophotometrically.

two ug of RNA from each and every sample was additional to a total volume of 25 ul response mixture con taining 2. 5 uM of oligo primer, and 200U of Molony Murine Leukemia Virus Reverse Transcriptase. The reac tion was initiated by incubating the reaction mixture for one h at 42 C for reverse transcription, and stopped by heating for ten min at 70 C. Aliquot of the just about every reverse transcription products was extra to your twenty ul reaction mixture containing LightCycler FastStart DNA Master SYBR Green І, 0. five uM of each primer corre sponding to mouse iNOS, Nox two, MKP one or b actin, and four mM MgCl2 to amplify the genes inside a LightCycler. The primer sequences used in this examine are listed in Table 1. Soon after pre incubation at 95 C for ten min, the polymerase chain reaction was carried out as follows 35 cycles of denaturation at 95 C for 15s, annealing at 60 C for 5s, and elongation at 72 C for 12s. two strategy was used to compare the mRNA expression amounts of genes in experimental groups with control groups.


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MessagePosté le: Mar 21 Oct - 07:41 (2014) Sujet du message: Publicité

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