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Our lab is interested in studying the interactions of macrophages with gram positive and gram negative bacteria. As part of these studies we perform live cell imaging of macrophages ingesting these bacteria. We use a vital nuclear stain, NucBlue, for these studies and have generated high quality images as a result.
Nuclear Staining for Live Cell Imaging
J774A.1 murine (BALB/c) macrophage cell line, Gram negative and Gram positive bacteria for bacterial infections
For live cell imaging, the J774A.1 murine (BALB/c) macrophage cell line was grown at 37C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum and 2mM L-Glutamine. J774A.1 cells were seeded in 6-well plates (Nunc) 18h before experiments (2 ml medium/well). Two drops of NucBlue (Life-Technologies) were applied to each well. Plates containing cells and NucBlue were placed in a humidity-and temperature-controlled environment. Thirty minutes after staining, the medium was replaced in a single step with 2 ml of fresh culture medium containing Gram-positive or Gram-negative pathogenic bacteria. Time-lapse fluorescence microscopy was carried out on a Nikon Eclipse Ti-E microscope (Nikon), equipped with a PlanFluor 40x 0.6NA objective (Nikon) and a CO2 incubator. Images were collected from every 2 min with an ORCA- R2 CCD camera (Hamamatsu) powered by Nis Elements 3.2 software. For NucBlue, a 375-390-nm excitation, 420-490-nm emission filter was used. For immunofluorescence experiments on fixed cells, macrophages were placed in 24-well tissue culture plates containing round glass coverslips. Phagocytic cells were infected with a Gram negative bacterium for 3h. After infections, cells were washed and fixed with cold paraformaldehyde (3.2% in PBS) for 20 min at room temperature. After being fixed, the cells were permeabilized with Triton X-100 (0.1% in PBS) for 5 min at room temperature (RT), and washed four times with PBS. Atto-488 phalloidin (Sigma), which binds polymerized F-actin, was used to identify actin filaments and fibers. NucBlue was diluted from 1:1 to 1:100.000 with BSA-PBS, and cells were stained during 25 mins at RT. Coverslips were mounted on glass slides with ProLong (Life technologies). All preparations were examined by epifluorescence microscopy using a Zeiss Axiovert 200 Microscope. Digital images were acquired using a Zeiss AxioCamHRc digital camera and merged using Photoshop CS3 (Adobe) software.
For short experiments, only one drop/ml of NucBlue was enough for 1 ml cell culture medium, which results in a good fluorescence signal. For immunofluorescence on fixed cells, NucBlue can be used at high dilution ratios.
When we studied the nuclei of macrophages by NucBlue fluorescence staining, we observed an excellent nuclear staining with very good signal, and low background. Optimization depends on cell type, and experimental setup. We use a 1:10.000 dilution for most experiments on fixed cells.
For immunofluorescence experiments, the fixation method should be selected on the basis of the experimental requirements. For live-cell imaging, NucBlue is a very good dye for nucleic acids on live cells, and did not require cold storage. Fixing cultured cells in paraformaldehyde results in excellent staining with NucBlue. In addition, this dye stains very well also bacterial DNA on fixed samples (not shown).
Image Caption
A) Upper panel. Live-cell imaging.
Cells were infected with a pathogenic bacterium and observed through time-lapse. Cells were monitored every 2 minutes with a ×40 PlanFluor objective. Merged images of the phase contrast and fluorescence are shown. The NucBlue (nuclei) signal is shown in blue. a) is at 5.42 min; b), 60.03 min; c), 102.58 min and d), 147.98 min. Arrows indicate apoptotic cells.
B) Lower panel. Immunofluorescence labeling on fixed cells.
The panel shows merged immunofluorescence images of macrophages infected with a Gram negative bacterium. In the merged images, the actin cytoskeleton is shown in green. NucBlue-stained nuclei are shown in blue. NucBlue dilutions on BSA-PBS are indicated on the left. Original magnification: ×200. Bars indicate 20mm.
Easy to use. Very good performance on fixed cells even at high dilution ratios.
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NucBlue has worked well for us for both live cell and fixed cell imaging
Product Name: NucBlue® Live ReadyProbes™ ReagentSupplier Page