Man2, a1 and Ricin Resistance in ES Cells Figure 6. Anti-ricin immunogold staining of either NN5 or ricin resistant F10 cells. A–C, images showing ricin particles located in various sites within NN5 cells treated with 20 pM ricin for 1 h at 37uC. D and E ricin-resistant F10 cells exposed to 20 pM ricin and processed for anti-ricin staining at the same time. NM, PM, Endo, OM and IM represent nuclear membrane, plasma membrane, endosome, outer membrane and inner membrane respectively (small arrows). The nucleus, cytoplasm and Golgi are also indicated. doi:10.1371/journal.pone.0022993.g006 Isolation of ricin resistant clones Libraries of NGG5.3 cells with gene-trap mutations (106 cells per 90 mm plate of feeder cells) were exposed to 20 pM ricin for 3 days; the plate was rinsed and maintained with normal medium over the next 7 days. Resistant colonies were grown until they were visible and picked into 96 well feeder plates. Neutral red and methylene blue assay for determining cell viability NN5, AB2.2, NGG5.3 and F10 ricin resistant cells were plated at 105 cells per well in 24 well plates and exposed to a range of ricin concentrations for 48 h. The cells were then mixed with 0.3% neutral red, incubated for a further 1 h and washed gently with phosphate buffered saline (PBS) for 1 h. After removal of the PBS, remaining viable cells were solubilised in a 1% glacial acetic acid/49% H20/50% ethanol solubilisation buffer and values determined at OD 490 nm. As an alternative method, viable cells were directly stained in the plates with 2% methylene blue in 70% ethanol for 10 minutes and then washed extensively in tap water. Isolation of the transposon–chromosome junction Isolation of the transposon–chromosome junction was per- formed using the splinkerette-PCR method as described [28]. The protocol was modified for use with ES cells on 96- or 24-well plates. Briefly, genomic DNA was isolated from ES cell colonies on 96- or 24-well plates; 2–4 mg DNA was digested with Sau3AI and ligated with the corresponding splinkerette adaptors HMSp– Sau3AI (generated by annealing splinkerette oligos HMSpBb– Sau3AI with HMSpAa). A first round of PCR was carried out with splinkerette primer HMSp1 and PB transposon primers PB59-1 or PB39-1. Then 1% of the PCR product was directly used for a second-round nested PCR, which was carried out with splinkerette primer HMSp2 and the PB transposon primers PB59- 2 or PB39-2. Primers for splinkerette-PCR and sequencing are shown in Supplementary table S1. The nested PCR products were purified by High Pure 96 UF cleanup plate (Roche) and were used for sequencing with primer PB59-seq or PB39-seq separately. Southern blotting and hybridization Total genomic DNA was restricted; size fractionated on agarose gels, blotted, and hybridized using standard procedures. The following probes were used: LacZ probe, a 800 base pair ClaI digested fragment from pSAgeo [14], which is a plasmid containing the SA-b-geo cassette in pBlueScript. PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e22993