Journal: Scientific Reports

Article Title: Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging

doi: 10.1038/s41598-017-07820-6

Figure Lengend Snippet: Schematic of the structure of Pink Flamindo. ( a ) Drawings for mApple and resultant Pink Flamindo. Asterisks indicate mutations. ( b ) 3D representation of Pink Flamindo bound (right) and unbound (left) to cAMP. Images were created using structural graphics from mCherry (PDB_4ZIO), which has the same origin of gene as mApple and Epac1 (cAMP-unbound: PDB_2BYV, cAMP-bound: PDB_4MGK).

Article Snippet: The adeno-associated virus (AAV) pAAV-hGFAP-Pink Flamindo and pAAV-hGFAP-Flamindo2 constructs were synthesised by replacing the promoter and coding region of the pAAV-hSyn-EGFP vector (Addgene plasmid #50465, a gift from Bryan Roth) with the human GFAP promoter and Pink Flamindo or Flamindo2 sequence; the latter was inserted between the Age I and Not I sites.

Techniques:

Journal: Scientific Reports

Article Title: Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging

doi: 10.1038/s41598-017-07820-6

Figure Lengend Snippet: Spectroscopic characterisation of Pink Flamindo. ( a ) Excitation and emission spectra of purified Pink Flamindo protein in the presence (solid line) or absence (dashed line) of 100 μM cAMP. Each fluorescent intensity (FI) was normalised to the peak of FI in the absence of cAMP. ( b ) Absorbance spectra of 30 μM Pink Flamindo in the presence (solid line) or absence (dashed line) of 100 μM cAMP. ( c ) Dose-response curve of Pink Flamindo for cAMP (●, red line) and cGMP (■, gray line). K d values calculated using the Hill equation were 7.2 μM for cAMP and 94 μM for cGMP. The peak of FI in the absence of cAMP or cGMP was normalised to 0, and the peak of FI in the presence of 1 mM cAMP was normalised to 1. The data represent the means ± standard deviation (n = 3). ( d ) Titration curves of Pink Flamindo against pH in the presence (●, solid line) or absence (○, dashed line) of 100 μM cAMP. The peak FI for each pH was normalised to the peak of FI in the presence of cAMP at pH 9.0. The data represent the means ± standard deviation (n = 3).

Article Snippet: The adeno-associated virus (AAV) pAAV-hGFAP-Pink Flamindo and pAAV-hGFAP-Flamindo2 constructs were synthesised by replacing the promoter and coding region of the pAAV-hSyn-EGFP vector (Addgene plasmid #50465, a gift from Bryan Roth) with the human GFAP promoter and Pink Flamindo or Flamindo2 sequence; the latter was inserted between the Age I and Not I sites.

Techniques: Purification, Standard Deviation, Titration

Journal: Scientific Reports

Article Title: Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging

doi: 10.1038/s41598-017-07820-6

Figure Lengend Snippet: Live cell imaging using Pink Flamindo with photoactivated adenylyl cyclase, and dual-colour imaging with G-GECO. ( a ) Sequential images and time course of fluorescence intensity of Pink Flamindo expressed in HeLa cells after consecutive exposure to 100 μM Fsk, 200 μM IBMX and 100 μM DDA. Scale bar represents 20 μm. The data represent the means ± standard deviation (n = 30 cells from 5 experiments). ( b ) Sequential images and time course of fluorescence intensity of MIN6 m9 cells expressing Pink Flamindo during the application of 25 mM glucose. Scale bar represents 20 μm. The data represent the means ± standard deviation (n = 15 cells from 4 experiments). ( c ) Sequential images and time course of fluorescence intensity of HeLa cells co-overexpressing Pink Flamindo and photoactivated adenylyl cyclase (bPAC) upon blue light laser excitation at 1.8 μW. Scale bar represents 20 μm. The data represent the mean ± standard deviation (n = 23 cells from 4 experiments). ( d ) Sequential images of MIN6 m9 cells co-overexpressing Pink Flamindo and G-GECO after consecutive exposure to 200 μM tolbutamide. Scale bar represents 20 μm. ( e ) Time course of fluorescence intensity of MIN6 m9 cells co-overexpressing Pink Flamindo and G-GECO. The data represent means ± standard deviation (n = 18 cells from 4 experiments).

Article Snippet: The adeno-associated virus (AAV) pAAV-hGFAP-Pink Flamindo and pAAV-hGFAP-Flamindo2 constructs were synthesised by replacing the promoter and coding region of the pAAV-hSyn-EGFP vector (Addgene plasmid #50465, a gift from Bryan Roth) with the human GFAP promoter and Pink Flamindo or Flamindo2 sequence; the latter was inserted between the Age I and Not I sites.

Techniques: Live Cell Imaging, Imaging, Fluorescence, Standard Deviation, Expressing

Journal: Scientific Reports

Article Title: Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging

doi: 10.1038/s41598-017-07820-6

Figure Lengend Snippet: In vivo imaging of cerebral cortical astrocytes using Pink Flamindo. ( a ) Image showing the experimental setup of a mouse under anaesthesia, positioned under a two-photon microscope. ( b ) Schematic of the cranial window for in vivo two-photon imaging. ( c ) Sequential images of cortical astrocytes expressing Pink Flamindo during exposure to 50 μM Fsk and 500 μM IBMX. Scale bar represents 100 μm. ( d ) Enlarged image of c (24 min) to indicate the regions of interest. Analysed cells are marked in red. Yellow dashed line indicates the coverslip border (see b ). Scale bar represents 50 μm. ( e ) Population mean trace of fluorescence intensity for Pink Flamindo from the images shown in c . The topical application of Fsk, IBMX and compound washout (indicated by black horizontal bars) was repeated twice. The data represent the means ± standard deviation (n = 9 cells).

Article Snippet: The adeno-associated virus (AAV) pAAV-hGFAP-Pink Flamindo and pAAV-hGFAP-Flamindo2 constructs were synthesised by replacing the promoter and coding region of the pAAV-hSyn-EGFP vector (Addgene plasmid #50465, a gift from Bryan Roth) with the human GFAP promoter and Pink Flamindo or Flamindo2 sequence; the latter was inserted between the Age I and Not I sites.

Techniques: In Vivo Imaging, Microscopy, In Vivo, Imaging, Expressing, Fluorescence, Standard Deviation

Journal: iScience

Article Title: Red blood cell signaling is functionally conserved in Plasmodium invasion

doi: 10.1016/j.isci.2024.111052

Figure Lengend Snippet: RH5-basigin interaction triggers a rise in cytosolic cAMP level Recombinant rRH5 was purified and used for all the assays. (A) cAMP immunoassay of RBCs was carried out. The dynamic changes of RBC cAMP level were measured in the absence (RBC only) and presence of 0.2 mg/mL rRH5 protein (RH5) within 10 min in 2-min intervals. Forskolin was used as a positive control (Forskolin). Experimental data presented as mean ± s.e.m, n = 3. ∗∗∗ p = 0.0006 indicates the significant difference on the cAMP level at 2 min compared to that at 10 min. (B) cAMP inhibition immunoassay of RBCs. The dynamics of RBC cAMP level was measured upon rRH5 binding for 2 min in the absence (RH5) and presence of anti-basigin Fab fragment (Fab+rRH5), G protein inhibitor BIM46187 (BIM46187+rRH5) or AC inhibitor 2′,5′-ddA (2′,5′-ddA + rRH5). Forskolin was used as a positive control (Forskolin). The cAMP reading in RBC alone was used as a negative control. The fold change in cAMP level in the samples was compared to that in the negative control and plotted as a bar chart. $$$ p = 0.002, ∗∗∗ p = 0.003 and ### p = 0.002 indicate the significant difference on fold change in RBC cAMP level upon rHR5 binding between in the absence (rRH5) and presence of anti-basigin Fab fragment (Fab+rRH5), BIM46187 (BIM46187+ rRH5) or 2′,5′-ddA (2′,5′-ddA + rRH5), respectively. Experimental data presented as mean ± s.e.m, n = 3. (C) Fluorescence images of RBC cAMP. Pink Flamindo loaded rRBCs were incubated in the absence (i, RBC only) and presence of forskolin (ii, Forskolin), rRH5 (iii, rRH5), anti-basigin Fab fragment (iv, Fab+rRH5), BIM46187 (v, BIM46187+rRH5), 2′,5′-ddA (vi, 2′,5′-ddA + rRH5), PKA (14–22) (vii, PKA (14–22)+rRH5) or verapamil (viii, Verapamil+rRH5) prior to rRH5 binding for 10 min. Fluorescence (left) and bright field (right) images were taken under 100× objective lens. Scale bars = 5 μm. (D) RBC cAMP measurement by using fluorescence plate reader. The cAMP signal intensity of the samples obtained from (C) were also measured. Dynamics of RBC cytosolic cAMP level was measured over 600 s ΣΔF(t) which reflects the cumulative change in RBC cytosolic cAMP was plotted as a bar chart. ∗∗∗ p = 0.006, ### p = 0.007, and $$$ p = 0.012 indicate the significant difference in RBC cAMP level upon rHR5 binding between in the absence (rRH5) and presence of anti-basigin Fab fragment (Fab+rRH5), BIM46187 (BIM46187+ rRH5) or 2′,5′-ddA (2′,5′-ddA + rRH5), respectively. Whereas no significant inhibition effect was seen in PKA (14–22) or Verapamil with p > 0.05 (n.s.) including p = 0.22 and p = 0.24 (color in blue). Experimental data presented as mean ± s.e.m, n = 3. Statistical comparison was done using one-way ANOVA.

Article Snippet: Pink Flamindo cAMP reporter sequence was amplified from pcDNA3.1-Pink Flamindo (Addgene), containing a red fluorescent protein variant mApple from amino acid 1–236, in which two linker positions at amino acid 150 and 151 flanks a cAMP-binding domain derived from amino acid region 205–353 of the EPAC protein.

Techniques: Recombinant, Purification, Positive Control, Inhibition, Binding Assay, Negative Control, Fluorescence, Incubation, Comparison

Journal: iScience

Article Title: Red blood cell signaling is functionally conserved in Plasmodium invasion

doi: 10.1016/j.isci.2024.111052

Figure Lengend Snippet:

Article Snippet: Pink Flamindo cAMP reporter sequence was amplified from pcDNA3.1-Pink Flamindo (Addgene), containing a red fluorescent protein variant mApple from amino acid 1–236, in which two linker positions at amino acid 150 and 151 flanks a cAMP-binding domain derived from amino acid region 205–353 of the EPAC protein.

Techniques: Recombinant, Giemsa Stain, Saline, Lysis, Membrane, Fluorescence, Blocking Assay, Purification, Protein Purification, cAMP Assay, Competitive ELISA, Plasmid Preparation, Software, Imaging