Cells were seeded in 8-well slides (ibidi GmbH, μ-Slide 8-Well ibiTreat, 80826), washed, fixed with 4% PFA and permeabilized using 0.1% TritonX-100. After blocking, cells were incubated with anti-Foxo1 antibody and a secondary antibody (Alexa Fluor 488, A-11008, Invitrogen (Carlsbad, CA, USA); 1 h each). Nuclei were visualised using 5 μg ml−1 Hoechst33342. Microscopy was conducted using LSM 510 Meta (Zeiss, Jena, Germany) microscope.
μ slide 8 well ibitreat
Manufactured by Ibidi
Sourced in Germany
The μ-Slide 8-Well ibiTreat is a laboratory equipment product. It is a multi-well slide designed for cell culture and microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Matlab, by MathWorks (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Anti phospho histone h2a x ser139, by Merck Group (1 mentions)
Tcs spe, by Leica Microsystems (1 mentions)
Anti atm ps1981, by Rockland Immunochemicals (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Leica sp8 confocal microscope, by Leica Microsystems (1 mentions)
Axiovert 200m fluorescence microscope, by Zeiss (1 mentions)
Metamorph imaging software, by Universal Imaging (1 mentions)
6 protocols using μ slide 8 well ibitreat
1
Immunofluorescence Staining of Foxo1
2
Immunofluorescence Analysis of DNA Damage Response
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Human mesenchymal stem cells were plated on μ-Slide 8 well ibitreat (Ibidi, Martinsried, Germany), fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 and blocked with 6% (wt/vol) BSA (Sigma-Aldrich) and 2.5% (vol/vol) normal goat serum (Sigma-Aldrich). Cells were then stained with the following primary antibodies: mouse monoclonal antibodies anti-phospho-Histone H2AX(Ser139) 1:500 (clone JBW301; Millipore, Billerica, MA, USA) and anti-ATMpS1981 1:300 (clone 10H11.E12, Rockland) or rabbit polyclonal antibody against 53BP1 1:500 (Novus Biological, Cambridge, UK) for 2 hrs at 4°C. Secondary antibodies were: Alexa 488 or Alexa 546 conjugated goat anti mouse and Alexa 546 conjugated goat anti-rabbit (all 1:500; Molecular Probes Inc.). Nuclei were stained with 0.1 μg/ml 4′-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) and slides mounted by using Mowiol solution (Calbiochem). Fluorescence images were obtained with a TCS-SPE, Leica Microsystem at 63× magnification. γH2AX foci quantification was performed counting at least 300 cells for each time-point.
3
Imaging Lipid Droplets in Huh-7.5.1 Cells
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Huh-7.5.1 cells seeded in μ-Slide 8 Well IbiTreat (Ibidi, Madison, WI, USA) were fixed in 3% v/v paraformaldehyde in PBS, then permeabilized in PBS containing 0.01% digitonin or 0.1% Triton X-100. Cells were probed with primary rabbit anti-capsid (C) antibody [20 (link)] (1:1000, kindly provided by Dr. Andrea V. Gamarnik (Fundación Instituto Leloir-CONICET, Argentina)), then incubated with secondary antibodies (Alexa 647 conjugated goat anti-rabbit), Hoechst dye, and Nile red dye diluted in PBS. Nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is a selective fluorescent stain for detecting intracellular lipid droplets [38 (link)]. The wells were then imaged using Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) with a 100x objective (HC PL APO 100x/1.40 OIL). All quantified images were acquired using the same laser intensity and gain settings, and LDs were enumerated by applying the same threshold setting to each image. For the LD analysis based on Nile red fluorescence, the calculated values reported for the LD-positive area (μm2) per cell are means ± SEM (>50 cells analyzed) and the numbers of LDs per cell are means ± SEM (>50 cells analyzed).
4
Quantifying HIV-1 and HIV-2 RNA Packaging
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To determine HIV-1 RNA packaging efficiencies, 293T cells were transfected with 50 ng Gag-CeFP construct, 50 ng Gag (untagged) construct, and 1 μg Bgl-YFP using TransIT-LT1 (Mirus). To investigate whether there was a preference for packaging HIV-1 or HIV-2 RNA, 293T cells were transfected with 50 ng Gag-CeFP construct, 50 ng Gag (untagged) construct, 200 ng 2-fsGag-MSL, 1 μg Bgl-mCherry, and 1 μg MS2-YFP using TransIT-LT1. Virus-like particles were collected 20 h posttransfection, clarified through 0.45-μm-pore-size filters, and plated onto glass slides (μ-Slide 8-well ibiTreat; Ibidi) with Polybrene (50-μg/mL final concentration). The slides were centrifuged at 1,200 × g for 1 h at room temperature and imaged using fluorescence microscopy as previously described (69 (link), 70 (link)). Images were analyzed using custom MATLAB (MathWorks) programs to determine the percentage of CeFP+ particles that contained HIV-1 RNA and/or HIV-2 RNA. At least 1,000 Gag-CeFP+ particles were analyzed per sample for each biological replicate, with at least three replicates performed for each construct.
5
RNA Copackaging Frequency Determination
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As previously described (28 (link)), to determine RNA copackaging frequencies, vector pairs were cotransfected into 293T cells, along with the RNA-labeling plasmids MS2-YFP and Bgl-mCherry, using TransIT-LT1 (Mirus). Although only Gag-CeFP constructs are depicted in the Supplementary Figure 2 , plasmids encoding Gag-CeFP and untagged Gag were transfected at a 1:1 weight ratio in all experiments. Viral particles were collected 20 h post-transfection, clarified through 0.45-μm-pore-size filters, and added to glass slides (μ-Slide 8 Well ibiTreat; Ibidi) with polybrene (50 μg/ml final concentration). The slides were centrifuged at 1200 × g for 1 h at room temperature and imaged using fluorescence microscopy as described previously. Images were analyzed using custom MATLAB (MathWorks) programs to determine packaging efficiencies (i.e. the percentages of CeFP+ particles that were YFP+, mCherry+, or YFP+ and mCherry+). At least 1000 Gag-CeFP+ particles were analyzed per sample for each biological replicate, with at least three replicates performed for each experimental group.
6
Live-cell Imaging of RBMY and PIM1
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HCC cell lines (4 × 104 cell/well) seeded onto chambered coverslips (μ-slide 8-well ibitreat; Ibidi, Gräfelfing, Germany) were grown overnight at 37ºC. Five hours after transfection with pEGFP-RBMY and pAsRed2-PIM1, live cells were subjected to time-lapse digital fluorescent imaging. Images were captured at 15-minute intervals and recorded for 18 hours using an Axiovert 200M fluorescence microscope (Zeiss, Ltd, Hertfordshire, UK) equipped with a CoolSNAP HQ2 (link) CCD camera (Photometrics, Tucson, AZ) and under the control of MetaMorph imaging software (Universal Imaging Corp, Downingtown, PA).
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