Cacl2 mgcl2

HIS-NSG mice (male and female) were generated using the human stem cell neonate NSG protocol as previously described (18 (link), 19 (link)). In brief, human fetal liver was obtained from Advanced Bioscience Resources, California, USA. The tissue was mechanically cut into small pieces with surgical scissors and treated with 2 mg/mL collagenase D (Roche) in Hank's balanced salt solution with CaCl₂/MgCl₂ (Gibco) for 30 min in a 5% CO₂ at 37°C followed by filtering through 70-μm nylon cell strainers (BD Biosciences). CD34⁺ human hematopoietic stem and progenitor cells (HSCs) were isolated using the direct CD34 MicroBead kit (Miltenyi Biotec). One- to three day-old NSG mice were irradiated with 100 cGy and injected intrahepatically with 1–2 × 10⁵ CD34⁺ HPCs 24 h after irradiation. Some NSG mice received 50 μl of PBS as a control. The mice were bled 10–12 weeks after engraftment and peripheral lymphocytes were stained with monoclonal antibodies purchased from BioLegend: anti-mouse CD45 (clone 30-F11), anti-human CD45 (clone HI30), anti-human CD3 (clone UCHT1), anti-human CD4 (clone OKT4), anti-human CD8 (clone SK-1), and anti-human CD19 (clone HIB19) for 30 min at 4°C. After red blood cell lysis, the samples were analyzed by flow cytometry using a BD LSR II to assess reconstitution of the human immune system and visualized using FlowJo software.

MCF10A-BiAD cells were seeded sparse or confluent in an appropriate vessel. Medium was changed 24 h later to DMEM medium without calcium and magnesium (-CaCl₂ - MgCl₂, Gibco) for calcium depletion. To remove residual calcium, the cells were washed once with PBS without calcium and magnesium. As a control, DMEM medium containing CaCl₂ and MgCl₂ (Gibco) was used. Analysis via immunofluorescence or MSRE-qPCR was done 24 h after medium change.