, 1990; Timenetsky et al, 2006) Here, we show

, 1990; Timenetsky et al., 2006). Here, we show Torin 1 clinical trial for the first time that

contamination of SH-SY5Y cells by a strain of M. hyorhinis results in increased levels of calpastatin. The mycoplasma-infected cells exhibit lower calpain activation and diminished calpain-promoted proteolysis, compared with the noninfected (clean) cells. These findings have implications for studies on mycoplasma-contaminated cultured cells, and may be relevant to the role of mycoplasmas in some diseases. For the detection of mycoplasma contamination of the SH-SY5Y cell cultures, the EZ-PCR Mycoplasma Test Kit (Biological Industries, Israel) was used according to the manufacturer’s instructions. For the specific identification of the contaminating Mycoplasma species, PCR was performed using the primers 1623F – ACACCATGGGAG(C/T)TGGTAAT and 1623R – CTTC(A/T)TCGACTT(C/T)CAGACCCAAGGCAT, designed to amplify the variable spacer between the conserved 23s and 16s rRNA mycoplasma genes. The PCR product was isolated, sequenced and analyzed using the program blastn

of the National Center for Biotechnology Information with the nucleotide collection database (nr). The contaminating Mycoplasma species was grown in a modified Chanock medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (Biological Industries), as described previously (Yavlovich et al., 2004). The medium was inoculated with 1–5% of a frozen culture and incubated at 37 °C for 48–96 h. Cells were harvested at the late find more exponential phase of growth (pH 5.9–6.2) by centrifugation at 12 000 g for 15 min, washed and suspended in a solution containing 250 mM NaCl and 10 mM Tris-HCl (pH 7.4). Mycoplasma-free SH-SY5Y cells (obtained from Dr Talia Han, Kaplan Medical Center, Rehovot, Israel) were grown in RPMI-1640

supplemented with 2 mM l-glutamine, 10% FCS, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin (pen-strep solution) [growth medium (GM)] in 25-cm2 plastic culture flasks. Cells were induced to differentiate, by plating 1–2 × 106 cells in 60-mm Petri dishes, and cultured for 7 days in Dulbecco’s modified Eagle’s medium, Prostatic acid phosphatase supplemented with 2 mM l-glutamine, 10% FCS and pen-strep solution [differentiation medium (DM)], in the presence of 20 μM all-trans retinoic acid (Sigma, St. Louis, MO). The DM and retinoic acid were replenished every 48 h during the differentiation. Cultures were routinely checked (every 3–4 weeks) by PCR, as described above, to ensure that they were uncontaminated (clean). Clean SH-SY5Y cells, cultured in GM, were infected with the mycoplasma (isolated from the original contaminated SH-SY5Y cells), at a multiplicity of infection of 50. The infected cells were differentiated under the same conditions described above for clean cells. To study the effects of Ca2+ on differentiated clean and infected cells, CaCl2 (Sigma) (100 mM stock solution in double-distilled water) and ionomycin (Calbiochem, La Jolla, CA) (0.

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