Visualizing Candida albicans Cell Wall Components

In order to determine the CW components, triple staining of C. albicans cells was performed. The mannans were stained using ConA dissolved in PBS containing 3% BSA, 1mM Ca²⁺, and Mn²⁺ and streptavidin conjugated with Alexa Fluor-568. Total chitin was visualized using CFW. The unmasked β-glucans were stained according to the protocol of Wagener et al. [42 (link)]. The C. albicans 24 h cultures were centrifuged and washed twice with PBS (4000× g, 5 min) and adjusted to A600 = 1 in PBS with 3% BSA. First, cells were incubated using 5 μg/mL of ConA for 1 h, 37 °C. The cells were washed twice with PBS and treated with 1:200 Alexa Fluor 568 conjugated streptavidin for 1 h, 37 °C. The cells were washed twice with PBS and 5 μg/mL Fc–hDectin-1 was added. After 1 h incubation, the cells were washed twice (4000× g, 5 min) and resuspended in PBS. The Fc-hDectin-1 treated cells were incubated with 1:250 Alexa Fluor 448-conjugated anti-human IgG Fc antibodies for 1 h, 4 °C. After that, the cells were washed twice as above, resuspended in PBS, and stained with CFW (0.025 mM) for 5 min, RT [43 (link)]. The cells were washed twice as above, resuspended in PBS, and concentrated. The observation of the preparations (at least 50 cells for each investigated strain) was performed using a super-resolution microscope (ZEISS Elyra 7 with Lattice SIM; Oberkochen, Germany).