We previously demonstrated that mast cells co cultured with astro

We previously demonstrated that mast cells co cultured with astrocytes are activated by CD40 CD40L interaction, and the activated mast cells induce release of mediators that participate in pathophysiology of sellectchem chronic neurodegenerative diseases Inhibitors,Modulators,Libraries like MS. However, the role of astrocytes activated in the co culture is not yet clarified. Therefore, we hypothesized that both cells are bi directionally activated in vitro and in vivo, and exam ined the signaling pathways and role for astrocytes in the co culture system and EAE model. We observed that cross talk between astrocytes and mast cells through CD40 CD40L produces inflammatory cytokines by Rho family GTPases, and the produced cytokines re activate astrocytes through cytokine receptor Jak1 2 and STAT1 on tyrosine701 signaling pathways.

Methods Cell Inhibitors,Modulators,Libraries culture U87 glioblastoma cell lines were obtained from Korea Cell Line Bank and grown in Dulbeccos modi fied Eagles medium sup plemented with 10% fetal bovine serum, 10 U ml penicillin and 10 ug ml strepto mycin at 37 C in a 5% CO2 atmosphere. HMC 1 cells were kindly pro vided by Dr. J. H. Butterfield. Cells were cultured in Iscoves modified Dulbeccos medium containing 10% FBS at 37 C in a 5% CO2 atmosphere. These culture conditions were designated as control medium. Preparation of primary brain astrocytes and bone marrow derived mast cells Primary brain astrocytes were isolated from the cerebral cortices of 1 day old BALB c mice as previously described. In brief, animals were sacrificed Inhibitors,Modulators,Libraries by decapitation, meninges were removed, and cortices were minced and gently dissociated in Hanks balanced salt solution.

Cells were supplemented with DMEM containing 5% FBS, transferred into 75 cm2 culture flasks, and incubated at 37 C in a humidified atmosphere of 95% air, 5% CO2. After 14 days of culturing, floating microglia was removed by shak ing the flask vigorously. More than 95% of cells were stained for astrocyte specific glial Inhibitors,Modulators,Libraries fibrillary acidic protein. Bone marrow cells were flushed from femurs and tibias of BALB c mice as described pre viously. Briefly, red blood cells were lysed using 0. 1 M NH4Cl, and the remaining cells were washed, resuspended, and cultured for 5 weeks in RPMI 1640 supplemented with 10% FBS and 50% WEHI 3B conditioned media which contained IL 3. BMMCs were collected onto object glasses by cytospin.

Cells were fixed in metha nol for Inhibitors,Modulators,Libraries 2 3 min, and then stained with May Gr��nwald solution for 15 min followed by Giemsa solution for 10 min and by washing with H2O, and then BMMCs were confirmed under microscope. certainly Purity of BMMCs was more than 95% of total cells. Co culture of astrocytes and mast cells U87 cells or primary brain astrocytes were grown in 75 cm2 flasks until confluent, and then HMC 1 cells or BMMC, respectively, were added to each astrocyte flask because mast cells are floating cells. The cells were co cultured for up to 24 h.

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