Fig 1: Higher sensitivity of TREM2 knockout (KO) microglia to ADP is driven by increased purinergic receptor expression.(A) Schematic highlighting key downstream Ca2+ signaling events triggered by ADP. Cytosolic Ca2+ response to ADP is determined by functional expression and activity of P2Y12 and P2Y13 receptors, IP3 receptors, endoplasmic reticulum (ER) store Ca2+ content, and store-operated Ca2+ entry (SOCE) regulated by STIM and Orai proteins. (B) Representative images (left panel) showing overlay of Fluo-4 (green) and Fura-red (red) channels in wild type (WT) (top) and TREM2 KO (bottom) induced pluripotent stem cell (iPSC)-microglia before and peak Ca2+ response after ADP addition in Ca2+-free buffer. Scale bar = 20 µm. Average trace showing Ca2+ response to ADP in Ca2+-free buffer (middle panel, 64–83 cells). Quantification of peak signal (right panel, n = 264–289 cells, four experiments, Mann–Whitney test). (C–E) Dose–response curves showing baseline-subtracted peak Ca2+ responses to ADP in Ca2+-free buffer (C), percent of ‘responding’ cells (D), and peak Ca2+ responses only in ‘responding’ cells (E). N = 84–474 WT cells and 70–468 TREM2 KO cells, 2–5 experiments. (F) RNA normalized read counts of P2Y12 and P2Y13 receptor expression from bulk RNA-sequencing of WT and TREM2 KO iPSC-microglia (n = 4, adjusted p-values from DESeq2). (G) Representative histogram (left panel) showing plasma membrane (PM) expression of P2Y12 receptor in WT and TREM2 KO microglia. Cells were stained with BV421-labeled anti-human P2Y12 receptor antibody. Isotype control is shown as dashed line. Right panel shows summary of median fluorescence intensity (MFI) of P2Y12 receptor-labeled cells (n = 10 samples each, Student’s t-test). (H) Ca2+ traces (left panel) showing response to 1 µM ADP in Ca2+-free buffer after 30 min pretreatment with a combination of P2Y12 receptor antagonist PSB 0739 (10 µM) and P2Y13 receptor antagonist MRS 2211 (10 µM). Summary of the peak Ca2+ response (right panel, n = 40–79 cells, two experiments, Mann–Whitney test). Data are mean ± SEM. p-Values indicated by ****p<0.0001. Figure 2—source data 1.Higher sensitivity of TREM2 knockout (KO) microglia to ADP is driven by increased purinergic receptor expression.In this dataset, the results of ADP stimulation in 0 Ca2+, dose curve of ADP in wild type (WT) and TREM2 KO, P2Y receptor expression, expression of key calcium signaling proteins, and inhibition of P2Y receptors are included.