Ab102505

Sections of native and decellularised zinc fixed tissue were subject to von Kossa staining. Briefly, sections of tissue were rehydrated in a graded alcohol series before staining in a 1% w/v silver nitrate (AnalarR) solution in water for 1 h on a Kenro Light Box (2 × 15 W bulbs). Following this the samples were washed in a 158 mM sodium thiosulphate pentahydrate (VWR) solution in water for 5 min before rinsing in water and counterstaining with eosin for 30 s. Calcium quantification was performed using a commercial kit (Abcam; AB102505). Samples of tissue for calcium quantification were lyophilised to a constant weight using a Modulo D freeze dryer (Thermo) before being hydrolysed in 6 M hydrochloric acid for 6 h at 120 °C, at 15 lb sqin⁻¹ and subsequent neutralization with 6 M sodium hydroxide. The samples were then assayed according to the manufacturer’s recommendations. Calcium content was quantified by interpolation of the absorption of the sample data from a known set of calcium standards (Abcam; AB102505).

For quantification of calcium content, calcium released into the supernatant was determined using the o-cresolphthalein complexone method as previously described [26 (link)], using the Calcium Colorimetric Assay (ab102505; Abcam, Cambridge, UK) and normalized to total protein content.

Quantification of the calcium concentration in the supernatant of human bone marrow aspirates, collected in heparin, was performed using a calorimetric calcium assay kit (ab102505, Abcam, Cambridge, UK) following the manufacturer’s instruction. The human samples were taken at diagnosis and the murine samples, when the mice were moribund and sacrificed.
In murine samples, bone marrow plasma was obtained by flushing a femur in 300 µl of PBS and collecting the supernatant by centrifugation (250 × g for 10 min). The same kit was used for the human samples.
K562 and THP1 cell culture media were collected for calcium release measurement using the same calorimetric calcium assay kit as used for the bone marrow supernatant. 5 × 10³ cells were seeded and culture media was collected over 7 days.