Perfection v750 pro scanner

The colony forming unit (CFU) assay was used as an indicator of self-renewal potential of the hMSCs and the CFU-osteoblast (CFU-Ob) assay was used as an indicator of their osteogenic potential. Two million MNCs were seeded in duplicate in T25 culture flasks and grown in GM for the first 7 days, followed by transition to mineralization media for further 7 days. The mineralization media consisted of GM containing 0.01 M β-glycerophosphate (BGP, Sigma – Cat. No.: G9422) and 10^-8 M Dexamethasone (Dex, Sigma – Cat. No.: D8893). At day 14, the cultures were fixed with 10% formalin for 15 min at ambient temperature, after which alkaline phosphatase (ALP) positive colonies were stained using the Leukocyte Alkaline Phosphatase Kit -ALP (Sigma – Cat. No.: 85 L2) following the manufacturer’s instructions. Subsequently, colonies were stained using 0.5% Coomassie Brilliant Blue staining (Fluka – Cat. No.: 27815) solution for 10 minutes and images of the stained colonies were acquired using an Epson Perfection V750 PRO scanner. The total number of CFUs and ALP positive colonies was quantified using ImageJ 1.45 s software and the percentage of ALP positive CFUs calculated.

Limiting dilution cloning was performed as previously described [49 (link)]. In brief, synchronous ring stage parasite cultures were diluted with fresh PCM and RBCs to a haematocrit of 0.75% and a parasitaemia of 0.0006% (= parasite cell suspension). Each well of a flat-bottom 96-well microplate (Costar #3596) was filled with 200 μL PCM/0.75% haematocrit (= RBC suspension). In each well of row A, 100 μL of the parasite cell suspension was mixed with the 200 μL RBC suspension (1/3 dilution), resulting in a parasitaemia of 0.0002%, which equals approximately 30 parasites per well. Subsequently, 100 μL of the row A parasite cell suspensions were mixed with 200 μL RBC suspension in the wells of row B resulting again in a 1/3 dilution (approximately 10 parasites/well). This serial dilution was continued until the last row of the plate was reached. The 96-well microplate was kept in a gassed airtight container at 37°C for 11 to 14 days without medium change. Subsequently, using the Perfection V750 Pro scanner (Epson, Nagano, Japan), the 96-well microplate was imaged to visualise plaques in the RBC layer. The content of wells containing a single plaque was then transferred individually into 5-mL cell culture plates and cultured using PCM until a stably growing parasite culture was obtained.

The two E12-plastinated AT-calcaneus-PF complexes used in this study were taken from one entirely plastinated cadaver. The cadaver was embalmed according to Xu et al.¹⁵ . The plastinates were scanned at 1200 dpi (Epson Perfection V750 Pro Scanner, Epson, Jakarta, Indonesia). For histological analyses, one fresh AT-calcaneus-PF complex was embedded into paraffin and horizontally sectioned with a section thickness of 20 µm using the same orientation as for the plastinates. Subsequently, the selected sections that corresponded to the plastinated slices were stained with hematoxylin eosin (H&E) and Masson–Goldner as trichromic staining (all consumables by Dr. Hollborn GmbH & Co KG, Leipzig, Germany) and photographed using a Zeiss Axioskop 40 (Carl Zeiss AG, Oberkochen, Germany) combined with an Olympus DP22 camera system (Olympus K.K., Shinjuku, Japan).

Whole cell extracts prepared by glass bead extraction in 10% trichloroacetic acid were separated in SDS‐8% polyacrylamide gels and transferred to PVDF membranes as described (Matos et al,2008). Membranes were horizontally cut into 2–3 slices, which were incubated with primary antibodies for 2 h. Rabbit antibodies were used for detection of Ama1 (Oelschlaegel et al,2005; 1:2,000), Ase1 (a gift from David Pellman; Juang et al,1997; 1:1,000), Cdc20 (Camasses et al,2003; 1:2,000), Clb3 (Schwickart et al,2004; 1:3,000), Ndt80 (a gift from Kirsten Benjamin; Benjamin et al,2003; 1:5,000), Pds1 (Katis et al,2010; 1:500), Sgo1 (a gift from Adam Rudner; Lianga et al,2013; 1:1,000), β‐tubulin/Tub2 (a gift from Wolfgang Seufert; used in Oelschlaegel et al,2005; 1:20,000), and Cdc5 (1:5,000), Dbf4 (1:5,000), Rec8 (1:10,000), and Spo13 (1:5,000) (Matos et al,2008). We used goat antibodies from Santa Cruz for Clb1 (sc‐7647; 1:300) and Clb4 (sc‐6702; 1:400) and mouse monoclonal antibodies from Invitrogen for Myc (9E10; 1:1,000) and Pgk1 (22C5D8; 1:40,000). HRP‐conjugated secondary antibodies were detected on X‐ray films by ECL (GE Healthcare). Films were digitized using an Epson Perfection V750 Pro scanner.