Agar pads were prepared in 96-well glass bottom plates (in Vitro Scientific) by depositing 120 μl molten VBM + Fe media (3 g l−1 trisodium citrate, 2 g l−1 citric acid, 10 g l−1 K2HPO4, 3.5 g l−1 NaNH4PO4•4H2O, 1 mM MgSO4, 18 μM FeSO4) and 1% agarose into each well. For pPilG-GFP or pPilJ-GFP strains, 0.02% arabinose and 50 μg ml−1 gent was added to the molten agar pad solution. Ten minutes after pouring, a single colony was stabbed into each well and the plate was incubated at 37°C for 4 h. The bacteria were visualized with a CSU-X1 spinning disk confocal on a Nikon Eclipse Ti inverted microscope with an Andor Clara digital camera using Plan Apo 1.49 N.A. 100× Oil TIRF objective. Images were collected using differential interference contrast, 488 nm laser, and acquired with NIS-Elements software 4.10 (Nikon).
Nis elements software 4
Manufactured by Nikon
NIS-Elements software 4.10 is an image analysis and processing software developed by Nikon. It provides tools for acquiring, analyzing, and managing digital images from Nikon microscopes and cameras.
Automatically generated - may contain errors
Lab products found in correlation
Csu x1, by Nikon (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Gateway lr cloning, by Thermo Fisher Scientific (1 mentions)
Plan apo 40x objective, by Nikon (1 mentions)
Pdonr zeo, by Thermo Fisher Scientific (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Qiazol, by Qiagen (1 mentions)
Csu x1, by Yokogawa (1 mentions)
Eclipse ti inverted, by Nikon (1 mentions)
Cfi apo 100, by Nikon (1 mentions)
4 protocols using nis elements software 4
1
Microscopic Imaging of Bacterial Pilus Expression
2
Imaging Chlamydia-Infected HeLa Cells
3
Episomal Expression of Hc WET1 in G217B
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The Hc WET1 coding sequence, 1035 bp of the ACT1 promoter (ACT1p), and 729 bp of the CATB terminator (CATBt) were amplified from G217B gDNA and assembled into the Gateway entry vector pDONR/Zeo (Life Technologies) using restriction enzymes to generate BAS1504. A vector control construct, BAS252, was generated identically except lacking the WET1 CDS. Using LR Gateway cloning (Life Technologies) each pDONR/Zeo entry vector was recombined into the Hc episomal expression vector pDG33 (pDG33 is a derivative of pWU55 [72 (link)] with Hc URA5 added for selection and made Gateway compatible). The episomally-maintained vector control (ACT1p –CATBt) and ACT1p –WET1 –CATBt constructs were electroporated into G217Bura5Δ as previously described [72 (link)]. Protein and RNA was isolated simultaneously from each 37°C or RT culture using Qiazol (Qiagen, Netherlands) following the manufacturer’s instructions.
Cell morphology of vector control and WET1-expressing cells was determined using differential interference contrast (DIC) microscopy with a Yokogawa CSU-X1 (Yokogawa, Tokyo, Japan) spinning disk confocal mounted on a Nikon Eclipse Ti inverted microscope (Nikon, Tokyo, Japan) with a PLAN APO 40X objective (Nikon) and an Andor Clara digital camera (Andor, Belfast UK). Images were acquired by and processed in NIS-Elements software 4.10 (Nikon).
Cell morphology of vector control and WET1-expressing cells was determined using differential interference contrast (DIC) microscopy with a Yokogawa CSU-X1 (Yokogawa, Tokyo, Japan) spinning disk confocal mounted on a Nikon Eclipse Ti inverted microscope (Nikon, Tokyo, Japan) with a PLAN APO 40X objective (Nikon) and an Andor Clara digital camera (Andor, Belfast UK). Images were acquired by and processed in NIS-Elements software 4.10 (Nikon).
4
Fluorescent Protein Imaging Protocol
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Fluorescent proteins were visualized with a CSU-X1 spinning disk confocal on a Nikon Eclipse Ti inverted microscope with an Andora Clara digital camera using CFI APO 100× oil TIRF objective. Images were collected using differential interference contrast (DIC), 488-nm excitation (GFP), and 561-nm excitation (RFP) and analyzed with NIS-Elements software 4.10 (Nikon) and Fiji [87 (link)].
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