Axiocam mrm rev 3 monochrome digital

To determine the lectin-binding capacity of the lectin-like domain of LGR1_Llp1, S. cerevisiae and C. albicans agglutination assays were performed as previously described42 (link) with minor modifications. Briefly, overnight-grown cultures of S. cerevisiae BY4741 or C. albicans SC5314 cells were washed and suspended in PBS to final concentration of a 1% w/v cell suspension. 50 μl of these cell suspensions was added to the wells of 96-well U-bottomed plates (Cellstar® 650180, Greiner bio-one) together with 50 μl of a 400 μg/ml FITC labelled lectin domain of LGR1_Llp1, obtaining a final lectin concentration of 200 μg/ml. Strains without any lectin domain were used for negative control. The plates were incubated at room temperature for 15 minutes while gently swirling. Finally, the strains were spotted on glass slides and visualized by epifluorescence microscopy at 400-fold using the Zeiss Axio Imager Z1 microscope equipped with an AxioCam MRm Rev.3 monochrome digital camera.

A yeast agglutination assay was performed using the protocol described previously (Pretzer et al.12 (link)) but with minor modifications. Briefly, overnight-grown cultures of Lactobacillus strains were washed, suspended in phosphate buffered saline (PBS, pH 7.2) to a final concentration of 1 × 10¹⁰ CFUs/ml. Similarly, overnight cultures of S. cerevisiae or C. albicans cells were washed and suspended in PBS so as to a make a 1% w/v cell suspension. For agglutination, 25 μl of bacterial suspension was added to 25 μl of PBS followed by 50 μl of yeast cells suspension in a 96-well U-bottom well sterile plate (Greiner bio-one). The mixtures were incubated for 10 min at RT with gentle shaking. To study inhibition, 25 μl of methyl-α-D-mannopyranoside (50 mM; Sigma-Aldrich) was added to the bacterial suspension instead of PBS. To examine agglutination, the mixtures were spotted on a slide and viewed under a phase-contrast microscope (400-fold magnification, Zeiss Axio Imager Z1 microscope equipped with an AxioCam MRm Rev.3 monochrome digital camera). Alternatively C. albicans and S. cerevisiae were incubated with 200 μg/ml of fluorescein isothiocyanate (FITC)-labeled lectin domain of Cmpg5300.05_29 alone and in combination with 25 μl of methyl-α-D-mannopyranoside (50 mM; Sigma-Aldrich). As a control, a lectin L-type domain of lectin-like protein 2 (Llp2) from L. rhamnosus GG was used.