Maternal vitaminDdeficiency found to increase lifetime diabetes risk in offspring WUSTLmedschool NatureComms
. After isolation, including CD11b selection with microbeads cells were resuspended in flow cytometry buffer , and >10cells were analyzed for each sample. Monocytes were incubated with 10 µg/ml of anti-mouse APC-CD45.1 or 10 µg/ml of anti-mouse PE-CD45.2 for 15 min on ice, then washed before flow cytometry with utilization of 5 µg/ml APC mouse IgG2a/K and 5 µg/ml of PE mouse IgG2a/K isotype controls.
Cells were acquired using FACStar Plus flow cytometer. Cell aggregates, dead, and cellular debris were excluded based on FSC/SSC. Batch analysis by Flow Jo version 9.6.2 was used for gating consistency and selection of positive populations. Unstained samples were used to control background autofluorescence signals.
. Briefly, minced tissue was digested in phenol red-free DMEM + 0.5% BSA + collagenase D + DnaseI for 30 min at 37 °C. Cells were then sieved through 70-µm cell strainers and centrifuged at 50×for 3 min at 4 °C to initially separate hepatocytes and non-parenchymal cells . The supernatant containing NPCs was collected and NPC suspension was centrifuged at 163for 7 min at 4 °C to pellet the NPCs.
An unmixing of the spectral data was performed using SpectroFlo software . For unbiased analysis, FCS files were processed using OMIQ analysis platform. Doublets, debris, and dead cells were excluded, and live CD45+ were downsampled to an equal number of cells from each mouse. Data from independent experiments were pooled , and uniform manifold approximation and projection was used to visualize the data.
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