BMDMs were plated at 3 × 105 cells per well in 24-well tissue culture plates and treated with 2400 IU ml−1 IFNβ 24 h before infection. Cells were infected with Δhly Lm or E. coli at an MOI of 1 or 1:200 respectively in RPMI. At 60 min p.i., all cells were washed three times with PBS+ and cultured in RPMI medium containing 10% FBS, 50 μg ml−1 gentamicin and IFNβ. At 5, 10, 15, 20, 25 and 30 min p.i., cells were fixed with 2.5% PFA. Differential antibody staining was carried out to stain extracellular bacteria before permeabilizing the cells to stain for total bacteria. Cells were imaged on a Quorum spinning disk confocal scan head (Leica DMI 6000 B inverted fluorescence microscope, Hamamatsu ORCA Flash 4 sCMOS and color camera) equipped with a ×63 objective. Phagocytosis was measured by calculating internalized bacteria (total bacteria minus extracellular bacteria) over the time course using Volocity 6 software.
Dmi 6000 b inverted fluorescence microscope
Manufactured by Hamamatsu Photonics
The DMI 6000 B is an inverted fluorescence microscope manufactured by Hamamatsu Photonics. The core function of this microscope is to enable the observation and analysis of fluorescently labeled samples in an inverted configuration, where the objective lens is positioned below the sample.
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Lab products found in correlation
Scmos orca flash 4, by Hamamatsu Photonics (2 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Foxo1 antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor conjugated antibody, by Thermo Fisher Scientific (1 mentions)
Af1700, by R&D Systems (1 mentions)
Mab4360, by Merck Group (1 mentions)
Mab4381, by Merck Group (1 mentions)
Rabbit monoclonal anti nanog, by Cell Signaling Technology (1 mentions)
Rabbit monoclonal anti sox2, by Cell Signaling Technology (1 mentions)
F1804, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using dmi 6000 b inverted fluorescence microscope
1
Bacterial Phagocytosis Kinetics Assay
2
Visualizing Lm Phagosomal Acidification
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BMDMs were plated at 1 × 105 cells per well in µ-Slide 8 Well IBIDI chambers (#80826) and treated with 2400 IU ml−1 IFNβ 24 h before infection. Cells were infected with GFP-Δhly Lm at an MOI of 5 in RPMI. At 60 min p.i., all cells were washed three times with PBS+ and cultured in RPMI medium containing 10% FBS, IFNβ and 50 μg ml−1 gentamicin. Cells were pulsed with cresyl violet (1 μM) for 5 min to label acidified compartments73 (link), and washed prior to imaging and replaced with RPMI medium containing 10% FBS and IFNβ. 4 h p.i., cells were imaged live on a Quorum spinning disk confocal scan head (Leica DMI6000B inverted fluorescence microscope, Hamamatsu ImagEM ×2 camera) equipped with a ×63 objective. Cresyl violet-positive bacteria were quantified using Volocity 6 software.
3
Immunostaining of Drosophila Fat Body
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Fat body was dissected from male and female wandering L3 larvae in cold PBS, pH 7.4. Dissected fat body was pooled (10–15 larvae/genotype) and fixed with 4% PFA PBS for 30 min at 4°C before permeabilization in PBST (PBS and 0.1% Triton X-100) at room temperature. The tissue was then blocked with 10% goat serum for 1 h at room temperature. Anti-dFOXO primary antibody (a gift from Dr. Pierre Leopold, Institut Curie, Paris, France; (Slaidina et al., 2009 (link))) was diluted 1:200 in PBST with 10% goat serum, and fat body was incubated overnight at 4°C. Following three 10 min washes in PBST, fat body was incubated in Alexa Fluor 488 secondary antibody in PBST (1:500) for 1 h at room temperature. The fat body was then incubated in phalloidin 568 for 30 min at room temperature, followed by three 10 min washes in PBST, with DAPI diluted (1:1000) in the second wash. Samples were mounted in 10 μL Pro-Long Diamond (Thermo Fisher Scientific) using self-adhesive reinforcement labels (Avery #32203) as spacers. Mounted samples were cured overnight at room temperature before sealing with nail polish. Images were acquired using a Quorum spinning disk confocal scan head (Leica DMI 6000 B inverted fluorescence microscope, Hamamatsu ORCA Flash 4 sCMOS and color camera) equipped with a 63× objective.
4
FOXO1 Nuclear Localization Quantification
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For HepG2 siRNA knockdown experiments, cells were seeded in 6 well plates for 24 h prior to siRNA transfection with RNAiMAX for 24 h. Cells were then reseeded in 24-well plates with coverslips for an additional 24 h. Cells were serum deprived for 3 h, fixed and immunostaining was conducted as previously described (Frendo-Cumbo et al., 2019 (link)). Briefly, cells were permeabilized and blocked in PBS containing 0.2% saponin (Calbiochem) and 10% normal goat serum (SS-PBS) for 30 min. Subsequently cells were incubated for 1 h with FOXO1 antibody (#2880; Cell Signaling) in SS-PBS, washed three times with PBS and incubated with secondary Alexa Fluor conjugated antibodies and phalloidin-565 (Invitrogen) for 1 h. Cells were washed three times with PBS and mounted in fluorescence mounting medium (Dako). Images were acquired using a Quorum spinning disk confocal scan head (Leica DMI 6000 B inverted fluorescence microscope, Hamamatsu ORCA Flash 4 sCMOS and color camera) equipped with a 63× objective. FOXO1 nuclear localization was quantified using ImageJ software. Briefly, the nuclear compartment was marked using DAPI and the cell border was determined using phalloidin. The cytosolic region was isolated by removing the DAPI-nuclear mask from the phalloidin-whole cell mask. FOXO1 fluorescence was then measured in the nuclear and cytosolic regions and a ratio was calculated.
5
Pluripotency Marker Immunostaining Protocol
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The cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) for 15 min, incubated with 0.1% Triton X-100/PBS for 15 min, and blocked in 5% BSA/PBS for 1 hour. The cells were then incubated with the following primary antibodies overnight at 4°C: mouse monoclonal anti–TRA-1-60 (1:100, MAB4360; Millipore), mouse monoclonal anti–TRA-1-81 (1:100, MAB4381; Millipore), mouse monoclonal anti–SSEA-4 (1:100, 330402; BioLegend), rabbit monoclonal anti-NANOG (1:1000, 5232; Cell Signaling Technology), rabbit monoclonal anti-OCT4 (1:1000, 5677; Cell Signaling Technology), rabbit monoclonal anti-SOX2 (1:1000, 5024; Cell Signaling Technology), rabbit monoclonal anti-DNMT3B (1:1000, 67259; Cell Signaling Technology), mouse monoclonal anti-FLAG (1:1000, F1804; Sigma-Aldrich), and goat polyclonal anti-GATA6 (1:500, AF1700; R&D Systems). The cells were then washed three times with PBS and incubated with appropriate fluorescently labeled Alexa Fluor secondary antibodies (1:1000; Invitrogen) for 1 hour. The cells were then washed three times with PBS, incubated with 4′,6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml; Molecular Probes) for 10 min, and washed once with PBS. Images were acquired using the Leica DMI6000B inverted fluorescence microscope equipped with the Hamamatsu ORCA-R2 charge-coupled device camera and analyzed with the Leica application suite advanced fluorescence software.
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