Ti u microscope

THP-1 derived DCs (1 × 10⁴) were allowed to adhere to poly-L-lysine-coated coverslips for 5 min by cytospin and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature (RT). After fixation, the cells were washed with PBS followed by incubation in blocking buffer (5% BSA in PBS) for 30 min at RT. Then, a DC-SIGN antibody was added in blocking buffer and incubated at 4 °C overnight. The cells were subsequently washed with PBS, followed by the addition of Alexa Fluor^® 488 conjugated secondary antibody diluted in the blocking buffer. The cells were incubated in the dark for 1 h at RT, then counterstained with DAPI for 5 min. The stained cells were washed with 1× PBS, mounted with ProLong Gold, and examined. Photographs were captured using a Nikon TiU microscope (Nikon Instruments Inc., Melville, NY, USA). For tissue slides: Slides were incubated in normal serum and BSA blocking step at room temperature for 20 min. After incubation with primary antibody overnight at 4 °C, slides were labeled with Alexa Fluor dye–conjugated secondary antibody and mounted with ProLong Gold (Invitrogen). Image Acquisition: Slides were examined and photographs were captured using a Nikon TiU microscope (Nikon Instruments Inc., Melville, NY, USA).

The increases in free intracellular calcium concentrations, [Ca²⁺]i, were determined as described elsewhere [14 (link)]. Briefly, TBC (1 × 10⁶ cells/well) were seeded onto Willico-Dish wells and incubated with Fura-2/AM at 1 µM for 30 min in the buffer (110 mM, NaCl; 5.5 mM, KCl; 25 mM, NaHCO₃; 0.8 mM, MgCl₂; 0.4 mM, KH₂PO₄; 0.33 mM, Na₂HPO₄; 20 mM, HEPES; 1.2 mM, CaCl₂) with a pH 7.4. The changes in [Ca²⁺]i were monitored using a Nikon microscope (TiU) equipped with EM-CCD (Luca-S) camera with S-fluor 40× immersion oil objective (Nikon, Tokyo, Japan). Changes in [Ca²⁺]i were expressed as F₃₄₀/F₃₈₀ ratio. All test molecules were added in small volumes with no interruptions in recordings.

HCT8 and HCT116 cells were cultured on WillCo-dish wells with a glass bottom and loaded with Fura-2/AM (1 μM) for 60 minutes at 37°C in loading buffer that contained: 110 mM NaCl; 5.4 mM KCl; 25 mM NaHCO₃; 0.8 mM MgCl₂; 0.4 mM KH₂PO₄; 20 mM HEPES-Na; 0.33 mM Na₂HPO₄; 1.2 mM CaCl₂, pH 7.4 as described by Dramane et al. [24 (link)]. The changes in intracellular Ca²⁺ (F340/F380) were monitored under a Nikon microscope (TiU) by using an S Fluor 40× oil immersion objective. The planes were taken at z intervals of 0.3 μm, and NIS-Elements software was used to deconvolve the images. The microscope was equipped with an EM-CCD (Lucas) camera for real-time recording of 16-bit digital images. The dual excitation fluorescence imaging system was used for studies of individual cells. The changes in intracellular Ca²⁺ were expressed as Δ ratio, which was calculated as the difference relative to the peak F340/F380 ratio. The data were summarized from the large number of individual cells (20–40 cells in a single run, with 3–9 identical experiments done in at least 3 cell preparations). For experiments conducted in the absence of external calcium (0% Ca²⁺), CaCl₂ was replaced by 1 mM EGTA in the buffer. All test molecules were added in small volumes with no interruption in recordings.