The A549 cells were seeded at 3 × 105 cells/well and co-transfected with 0.5 μg of pGP-CMV-GCaMP6f (Addgene, MA, USA) and CMV-R-GECO1.2 (Addgene, MA, USA) 1.5 μL of Lipofectamine (Invitrogen, Lipofectamine 2000, Carlsbad, USA) in 50 μL of Opti-MEM medium (Gibco, USA) at room temperature for 5 min. The DNA–lipid complex was mixed with the cells and the mixture incubated at 37 °C for 1 day. After confirming the presence of transfected cells, DEX was treated for 6, 12, and 24 h prior to determine calcium concentration by using an automated microscope (BioTek, Lionheart FX).
Pgp cmv gcamp6f
Manufactured by Addgene
Sourced in United States
The PGP-CMV-GCaMP6f is a plasmid that encodes the genetically encoded calcium indicator GCaMP6f under the control of a CMV promoter. GCaMP6f is a fluorescent protein-based sensor that exhibits increased fluorescence upon binding to calcium ions, allowing for the monitoring of calcium dynamics in living cells.
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Lab products found in correlation
Pcmv g cepia1er, by Addgene (3 mentions)
Pcmv cepia2mt, by Addgene (3 mentions)
Prl sv40 plasmid, by Promega (3 mentions)
Pcdf1 mcs2 ef1 puro expression vector, by System Biosciences (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Pcdna3.1 gfp, by Thermo Fisher Scientific (2 mentions)
Lionheart fx, by Agilent Technologies (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Paav ef1a fdio eyfp, by Addgene (1 mentions)
Paav syn flex rc chrimsonr tdtomato, by Addgene (1 mentions)
Plentiekar2g2, by Addgene (1 mentions)
Gw1 phred, by Addgene (1 mentions)
Ptriex rhoa flare, by Addgene (1 mentions)
Egfp p65, by Addgene (1 mentions)
Pmitotimer, by Addgene (1 mentions)
Mcherry lifeact 7, by Addgene (1 mentions)
Prk5 myc rhoa t19n, by Addgene (1 mentions)
8 protocols using pgp cmv gcamp6f
1
Quantifying Glucocorticoid-Induced Calcium Signaling
2
Generating Flp-Dependent GCaMP6f AAV
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We generated an Flp-dependent GCaMP6f AAV construct for photometry experiments. pAAV-Ef1a-fDIO-EYFP (Addgene #55641) was digested with Asc I and Nhe I to remove the EYFP complementary DNA (cDNA), and GCaMP6f cDNA was obtained from pGP-CMV-GCaMP6f (Addgene #40755) (76 (link)) digested with Not I and Bgl II restriction enzymes. After incompatible single-strand overhangs were filled in to generate blunt ends, the two restriction fragments were ligated to produce plasmid pAAV-Ef1a-fDIO-GCaMP6f. To generate pAAV-Ef1a-fDIO-tdTomato, a tdTomato cDNA was isolated from pRSET-tdTomato (provided by R. Tsien) and cloned into pAAV-Ef1a EYFP digested with Asc I and Nhe I. To generate pAAV-Ef1a-fDIO-ChrimsonR-tdTomato, a ChrimsonR-tdTomato fusion cDNA was isolated from pAAV-Syn-FLEX-rc[ChrimsonR-tdTomato] (Addgene #62723) and cloned into pAAV-Ef1a-fDIO EYFP digested as above. The viruses and titers that were used are summarized in table S2.
3
Measuring Cytosolic Ca2+ in Mast Cells
4
Comprehensive RhoA Signaling Toolkit
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Raichu-RhoA (provided by M. Matsuda [27 (link)]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-myc-RhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).
5
Genetically Encoded Fluorescent Indicators
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The genetically encoded fluorescent Ca2+ indicator GCaMP6f (Chen et al. 2013 (link)) DNA sequence was amplified by PCR from pGP-CMV-GCaMP6f (Addgene) with BamHI and HindIII at the 5′ and 3′ ends, respectively, and sub-cloned into the recombinant adeno-associated virus (rAAV) vector pAAV-6P-SEWB (Shevtsova et al. 2005 (link)), with the human synapsin-1 (SYN) promoter to generate the construct pAAV-SYN-GCaMP6f. The human glial fibrillary acidic protein (GFAP) promoter (Hirrlinger et al. 2009 (link)) was inserted with MluI and BamHI into pAAV-SYN-GCaMP6f, resulting in the pAAV-GFAP-GCaMP6f construct. Virus from pAAV-SYN-iGluSnFR (Marvin et al. 2013 (link)) was used to express the genetically encoded fluorescent glutamate indicator iGluSnFR in neurons.
6
Plasmid Construction for ATF6 and CRT Studies
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Plasmids expressing full length mouse ATF6α and ATF6β were constructed by inserting ATF6α and ATF6β cDNAs into the pCDF1-MCS2-EF1-Puro expression vector (System Biosciences, Palo Alto, CA, USA). Plasmids expressing the active and dominant-negative forms of human ATF6β, ATF6β(392) and ATF6β(392)∆AD, respectively, were constructed in pcDNA3.1(+), as previously described14 (link). pcDNA3.1(+) GFP was obtained from Invitrogen/Thermo Fisher Scientific (Waltham, MA, USA). CAT plasmids containing the mouse CRT promoter (pCC1, pCC3, and pCC5) were provided by Dr. Marek Michalak (University of Alberta)17 (link). Both of pCC1 and pCC3, but not pCC5, contain ERSE, a consensus of CCAATN9CCACG18 (link) (Fig. 3 A). Luciferase plasmids containing huCRT(wt) and huCRT(mut), with the two ERSEs mutated in the latter, and a plasmid containing the WT human GRP78 promoter (huGRP78) were constructed as previously described18 (link). The pRL-SV40 plasmid was obtained from Promega (Madison, WI, USA). Plasmids for Ca2+ imaging such as pCMV G-CEPIA1er, pGP-CMV-GCaMP6f and pCMV CEPIA2mt were obtained from Addgene (Watertown, MA, USA). Cells were transfected with each plasmid for 5 h using Lipofectamine 2000 (Life Technologies) and further incubated for 24–48 h. In our model, transfection efficiency was approximately 5% in primary neurons.
7
Plasmid Construction and Transfection for Investigating ER Stress Pathways
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Plasmids expressing full length mouse ATF6α and ATF6β were constructed by inserting ATF6α and ATF6β cDNAs into the pCDF1-MCS2-EF1-Puro expression vector (System Biosciences, Palo Alto, CA, USA). pcDNA3.1(+) GFP was obtained from Invitrogen/Thermo Fisher Scientific (Waltham, MA, USA). CAT plasmids containing the mouse CRT promoter (pCC1 and pCC3) were provided by Dr. Marek Michalak (University of Alberta) (Waser et al., 1997) . Both plasmids contain ERSE, a consensus of CCAATN9CCACG (Yoshida et al., 1998) (Figure 3A). Luciferase plasmids containing huCRT(wt) and huCRT(mut), with the two ERSEs mutated in the latter, and a plasmid containing the WT human GRP78 promoter (huGRP78) were constructed as previously described (Yoshida et al., 1998) . The pRL-SV40 plasmid was obtained from Promega (Madison, WI, USA). Plasmids for Ca 2+ imaging such as pCMV G-CEPIA1er, pGP-CMV-GCaMP6f and pCMV CEPIA2mt were obtained from Addgene (Watertown, MA, USA).
Cells were transfected with each plasmid for 5 h using Lipofectamine 2000 (Life Technologies) and further incubated for 24-48 h. In our model, transfection efficiency was approximately 5% in primary neurons.
Cells were transfected with each plasmid for 5 h using Lipofectamine 2000 (Life Technologies) and further incubated for 24-48 h. In our model, transfection efficiency was approximately 5% in primary neurons.
8
Plasmid Constructs for ATF6 Study
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Plasmids expressing full length mouse ATF6α and ATF6β were constructed by inserting ATF6α and ATF6β cDNAs into the pCDF1-MCS2-EF1-Puro expression vector (System Biosciences, Palo Alto, CA, USA). pcDNA3.1(+) GFP was obtained from Invitrogen/Thermo Fisher Scienti c (Waltham, MA, USA). CAT plasmids containing the mouse CRT promoter (pCC1, pCC3, and pCC5) were provided by Dr. Marek Michalak (University of Alberta) 17 (link) . Both of pCC1 and pCC3, but not pCC5, contain ERSE, a consensus of CCAATN 9 CCACG 18 (Figure 3A). Luciferase plasmids containing huCRT(wt) and huCRT(mut), with the two ERSEs mutated in the latter, and a plasmid containing the WT human GRP78 promoter (huGRP78) were constructed as previously described 18 . The pRL-SV40 plasmid was obtained from Promega (Madison, WI, USA). Plasmids for Ca 2+ imaging such as pCMV G-CEPIA1er, pGP-CMV-GCaMP6f and pCMV CEPIA2mt were obtained from Addgene (Watertown, MA, USA). Cells were transfected with each plasmid for 5 h using Lipofectamine 2000 (Life Technologies) and further incubated for 24-48 h. In our model, transfection e ciency was approximately 5% in primary neurons.
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