Mϕs were seeded at 1 × 105 cells/well on eight-chamber Lab-Tek II slides (Nunc, Roskilde, Denmark) for 2 h at 37°C, 5% CO2 in either CM or serum-free (SF) RPMI 1640 medium. Cells were washed with FACS staining buffer (FSB, 0.5% BSA in DPBS without Ca2+ and Mg2+) and stained with 2 mg FITC-conjugated anti-FcϕR (CD16/CD32) Abs (eBioscience, San Diego, CA, USA) for 1 h at 4°C. Cells were then washed with FSB, fixed for 20 min with 4% paraformaldehyde followed by another wash with FSB and mounted with vectashield (Vector Laboratories, Burlingame, CA, USA) containing DAPI. Slides were analyzed using a Nikon Eclipse TE300 fluorescent microscope equipped with Axio Cam HRm (Zeiss, Jena, Germany). For phase contrast microscopy analysis, Mϕs were seeded (5 × 104) on TC-treated 96-well plates for 16 h at 37°C, 5% CO2 and visualized as previously described.
Lab tek 2 slides
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, Germany
Lab-Tek II slides are microscope slides designed for cell culture and tissue preparation. They feature a removable chamber that allows for controlled culture conditions and easy media changes. The slides are made of durable polystyrene and are available in various well configurations to accommodate different experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam hrm, by Zeiss (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Ix81 zdc2 inverted microscope, by Olympus (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Orca er, by Hamamatsu Photonics (1 mentions)
N2 and b27, by Thermo Fisher Scientific (1 mentions)
Chamber slide system, by Thermo Fisher Scientific (1 mentions)
3 protocols using lab tek 2 slides
1
Macrophage Phenotype Characterization
2
Immunofluorescence Staining of iRBCs
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To prevent nonspecific staining, the diluted rabbit sera were adsorbed on fresh normal human O+ red blood cells. In order to explore proteins expressed at the membrane surface, unfixed iRBCs were used in liquid phase. Pellets (5 μl) of cultured iRBC were incubated for 45 min at RT with 100 μl of serum diluted 5 times in PBS supplemented with 2% BSA and 100 μg/ml Hoechst reagent. After washing with PBS supplemented with 2% BSA, antibodies were detected with Alexafluor 488 conjugated goat anti-rabbit IgG (Life Technologies). Immunofluorescence staining was analyzed with a direct microscope (BX51, Olympus, Rungis, France). Fluorescence imaging of the HEK293 clones expressing the CBP candidates as EYFP chimera was performed on Lab-Tek II slides (Nunc, Dutscher) after fixation (PFA 4%, 30 min 4 °C) using an Olympus IX81-ZDC2 inverted microscope equipped for Total Internal Reflection fluorescence (TIRF). Digital images were captured on a cooled charge-coupled device (CCD) camera (ORCA-ER, Hamamatsu, Massy, France).
3
Plating Spheres on Fibronectin Coated Slides
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Second generation spheres were plated onto fibronectin‐coated Lab‐Tek®II slides (Chamber Slide™ system, Nalge Nunc International) or onto polylysine/fibronectin‐coated 12‐mm diameter glass cover slides (Assistent, Germany) in 24‐well plates (Multiwell 3047,14‐mm diameter per well; Falcon, Becton Dickinson, Franklin Lakes, NJ) in DMEM/high‐glucose (4,500 mg/L dextrose) and F12 medium (mixed 1:1) supplemented with N2 and B27 (Invitrogen). Eighty percentage of the medium was replaced twice a week. Differentiated cells were analyzed after 12–14 days by immunocytochemistry.
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