R geco1

Transient transfections of HeLa cells were performed using PolyJet purchased from SignaGen (http://signagen.com/). Plasmids for GCaMP6f (fast version of GCaMP6), R-GECO1.2, jGCaMP7f, and LAR-GECO1.2 were obtained from Addgene, and the plasmids were used for transient transfection in HeLa cells. The respective genes were cloned into the T. gondii expression vector pCTH3 and pDHFRTubGFP for chloramphenicol and selection-less stable expression of GCaMP6f in tachyzoites, respectively. Thapsigargin, ionomycin, saponin, histamine, Zaprinast, and all other chemicals were obtained from Sigma. All plasmids used in this work are shown in Table S3.

For REX-GECO1 plasmid with a CMV promoter, template (REX-GECO1 in pTorPE^{11 (link)}) was cloned into a modified pcDNA3 plasmid by PCR as previously described^{10 (link)}. This vector was used in one-photon, two-photon imaging of HeLa cells and one-photon imaging of dissociated rat hippocampal neurons. For REX-GECO1 plasmid with a human synapsin I promoter, template (REX-GECO1 in pTorPE) was cloned into an AAV2 plasmid flanked by restriction sites BamH1 and HindIII by PCR using following primers: BamH1_fw (5′-GAGGATCCACCATGGTCGACTCATCACGTC-3′) and HindIII_rv (5′-GCGATGAAGCTTCTACTTCGCTGTCATCATTTGTACAAACTCTTCGTAGTTT-3′). For iGluSnFR plasmid with a human synapsin I promoter, iGluSnFR (Addgene plasmid 41732) was used as a template and cloned into an AAV2 plasmid flanked by restriction sites BamH1 and HindIII by PCR using following primers: BamH1_iGlu_fw (5′-CGAGGATCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTAC-3′) and HindIII_iGlu_rv (5′-CCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGTTAGAAGCTTCGATCC-3′). For GCaMP6s R-GECO1 and RCaMP1h plasmid (with a human synapsin I promoter) used in comparison with REX-GECO1 in rat hippocampal organotypic brain slices, GCaMP6s (Addgene plasmid 40753), R-GECO1 and RCaMP1h (Addgene plasmid 42874) were used as a template and cloned into the same AAV2 plasmid, respectively.