Cells were subsequently transferred to PCR tubes using a micropipette. A total of 44 Giardia cysts from patient samples that had previously been labeled with FITC labeled CWP-specific antibodies were learn more picked and placed on a 12 well microscope slide (Thermo Fisher Scientific, Sweden) in order to verify the specificity of the FK228 price single cell isolation method. Isolated single cysts, were evaluated by trained, independent microscopists using a Nikon Eclipse E400 Fluorescent microscope (Nikon, Tokyo, Japan). In addition, one of the wells of the slides was always used

as a negative control, here liquid was transferred from the fecal suspension onto one of the wells on the 12-well slides and analyzed. Such negative controls were also implemented in the PCR based assays. DNA extraction of single Giardia cysts and trophozoites Two different methods were evaluated for efficient extraction of DNA from single Giardia trophozoites in order to establish a sensitive enough method for the purpose of generating sequences from single cells, where ASH can properly be assessed; 1. Snap freezing/thawing of single trophozoites in 1XPBS at −80°C

SN-38 post-isolation. 2. DNA extraction of single trophozoites using DNAreleasy (NIPPON Genetics Europe, No LS02, Düren, Germany). Isolated single Giardia trophozoites were deposited in 2 μl drops of 1XPBS, transferred to PCR tubes containing 3 μl DNAreleasy and treated according to the manufacturer’s instructions (both the short and the long protocols provided by the manufacturer were assayed for extraction of DNA from single cysts). Subsequently, PCR reaction mixtures were added to the samples, to a final volume of 25 μl. PCR and sequencing of target genes for comparative analyses Nested PCR was performed on DNA from single cells and trophozoites following the same protocols that have been used on DNA from crude isolates, generating a 530 bp amplicon of the tpi gene and a 511 bp amplicon of the bg gene [22, 23]. Also, in order to verify the assemblages in the clinical samples Avelestat (AZD9668) as well as on all single

cysts from isolate Sweh207, assemblage A and assemblage B specific PCRs for the tpi locus were performed [10, 24]. PCR products were verified on 1.5% agarose gels stained with GelRed (Biotium, Hayward, CA, USA), proper amplicons were purified with Exo-SAP ITTM according to the manufacturer’s instructions (GE Healthcare, Uppsala, Sweden) and sequenced bi-directionally using the BIG DYE 3.1 sequencing kit (Applied Biosystems, La Jolla, CA, USA). Sequenced products were analyzed using the AB 9100 sequence reader (Applied Biosystems, La Jolla, CA, USA), and subsequently examined and aligned utilizing the BioEdit software (Ver. 7.0.5.). Sequences The sequences generated in this study were submitted to GenBank, with the following accession numbers [GenBank:JN579665-JN579676] (tpi sequence set), and [GenBank:JN579677-JN579688] (bg sequence set).