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4 protocols using memerald

1

Connexin-32 Overexpression and Cell Viability

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Carbenoxolone disodium salt (CBX; Sigma) was used as a gap junction inhibitor. Human connexin-32 plasmid (Cx32), C-terminally tagged with mEmerald, was obtained from Addgene (plasmid #54054) [48 (link)] while pcDNA 3.1 mock plasmid was purchased from Invitrogen. EVOS fluorescent microscope was used to verify the expression of Cx32 in cells (Additional file 3: Figure S3). Co-cultures of GS-293 and FSHR-293 cells were transiently transfected with Cx32 plasmid to determine the effect of connexin overexpression on cAMP transfer.
For determining cell viability after CBX treatment, co-cultures of GS-293 and FSHR-293 were preincubated with different concentrations of CBX (25, 50, 75 and 100 μM) for 2h in assay medium. Assay medium was then discarded, followed by washing with PBS. Cell viability was then assessed by CellTiter AQueous non-radioactive cell proliferation assay (Promega) following manufacturer’s instructions. Briefly, cells were incubated in DMEM complete medium (without phenol red) containing MTS/PMS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate] solution for 2h (37°C, 5% CO2). Absorbance was then read at 490 nm, which provides a measure of cellular viability. One-way analysis of variance (ANOVA) was used to determine statistical differences among different samples.
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2

Fluorescent Protein Transfection Protocol

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FusionRed-F-tractin has been previously described [3 (link)]. EGFP-F-tractin (no. 58473), mEmerald (no. 53975), LifeAct-mEmerald (no. 54148), mScarlet-I (no. 85044), and LifeAct-mScarlet-I (no. 85054) were obtained from Addgene (Watertown, MA). 1 μg of plasmid was used to transfect 750,000 cells in each well of a 6-well plate using Lipofectamine 2000 (5 μL; Thermo Fisher Scientific) in OptiMEM (400 μL; Thermo Fisher Scientific). After 30 min at room temperature, plasmid in Lipofectamine 2000/OptiMEM was then incubated with cells in complete media (2 mL) overnight.
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Fluorescent Protein-Tagged Receptor Constructs

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The mCherry (when co-transfected with SHB) and mEmerald (when co-transfected with VEGFR2) FAK (PTK2) constructs were purchased from Addgene (Watertown, MA, USA, Addgene.org; #55122 and #54241). The eGFP-SHB construct was generated by inserting the human SHB cDNA starting with the initiator ATG in frame downstream of the eGFP sequence with a linker in the pEGFP-N1 vector. The SHB cDNA included 137 nucleotides downstream of the stop codon. The linker sequence (underlined) between eGFP and SHB was TACAAGTCCATGGCC.
The mCherry-VEGFR2 pcDNA3.1 construct contained VEGFR2 cDNA inserted into the KpnI site followed by a linker, and in frame with the mCherry sequence. The linker sequence was GGTGGGAGTGGAGGTGGGAGTGGA. The wild type receptor construct was used as a template for the mutants. The KpnI-AgeI receptor fragment was replaced with the corresponding mutant fragments that were obtained from previously generated cDNAs [22 (link)]. All constructs were sequence verified.
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4

Plasmid Repository for Cellular Imaging

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The pMD2.G VSV-G (#12259), H2B-mCherry (#20972), H2B-Halo Tag (#91564), TfR-EGFP(#54278), mEmerald (#53976), and p-AMPK alpha2 K45R (#15992) plasmids were obtained from Addgene. The AP180-C plasmid was a gift from Julie G. Donaldson. The plasmids for CAAX-EGFP (Farn-119), mTagBFP2-C1 (CV-261), Mito-EGFP (Clon-109) were obtained from the Michael Davison collection. iGlucoSnFR.mRuby2 was a gift from Loren Looger at Janelia Research Campus.
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