Figure 3

Mycoplasma eradication restored Aβ accumulation in APP-transfected cells. (A) APP (first panel) and Aβ (second panel) levels in wild-type (wt-293) and APP-transfected (APP-293) HEK293 cells were analyzed by WB, as described before. APP-293 cells were cultured for one week in the absence (-MRA) or presence (+MRA) of Mycoplasma Removal Agent (MRA, MP Biomedicals, Inc). Conditioned medium of untreated and treated APP-293 cells was analyzed by PCR for the presence of mycoplasma genomic DNA (lower panel), as described above. Twenty-five picograms of synthetic Aβ1–40 (IBL-America) were loaded as a control (lane 4). (B) Determination of the mycoplasma species by restriction fragment polymorphism analysis of PCR products. The PCR product was digested with six different restriction endonucleases as indicated and separated on an agarose gel. The fragment pattern identifies the contaminant as M. hyorhinis. Briefly, a PCR reaction is performed with a mix of 9 primers to cover a range of mycoplasma species, including major contaminants of cell cultures. The 500–520 bp PCR products, depending on the species, are then subjected to several digests with restriction endonucleases to differentiate between 7 mycoplasma species, as previously described [14]. Cells were cultured without antibiotic for several days. 1 ml of supernatant and 1 ml of trypsinized cells were centrifuged for 10 min at 13,000 rpm and DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen). The PCR was carried out in 50 μl containing 0.2 mM of each deoxynucleotide, 0.2 μM of each primer (sequences provided in Ref. [17]), 1.5 mM MgCl₂. PCR conditions were as follows, 5 min denaturation at 95°, 35 cycles of 30 sec at 95°C, 30 sec annealing at 65°C and 1 min extension at 72°C followed by a final extension for 10 min. 5 μl of the PCR products were used for restriction digests in 20 μl of reaction volume for 3 hrs. The following enzymes were used: Pfl FI (isoschizomer of Asp I), Bam HI, Hae III, Hpa I, BstBI (isoschizomer of Sfu I) and Xba I. Fragments were analyzed on a 1.5% agarose gel.