Calcofluor white stain

HT-29 colon carcinoma cells were maintained in DMEM (Lonza) supplemented with 10% heat-inactivated Fetal Calf Serum (FCS)(Serana), 2 mM glutamine (Lonza), 100U penicillin and 100 μg streptomycin (Lonza) at 37 ℃ and 5% CO₂. Cells routinely tested negative for mycoplasma contamination. Cells were seeded at 3 × 10⁵ cells ml^-1 in a 48-well culture plate (VWR) 6 days prior to the assay. Confluent monolayers were washed with DMEM before exposing them to yeast. C. albicans was grown overnight in Sabouraud Dextrose Broth (Sigma)(37 ℃, 200 rpm). 25 × 10⁶ yeast cells ml^-1 were then incubated for 30 min in 0.5 mg mL^-1 calcofluor white stain (Merck, Amsterdam, the Netherlands) (37 ℃, 200 rpm), washed and resuspended at 8 × 10⁶ cells ml^-1 in DMEM without phenol red (Lonza). Next, HT-29 cells were exposed to 4 × 10⁵ yeast cells in 200 μl for 1 h at 37 ℃. Non-adhered cells were washed away and fluorescence intensity was measured on a Clariostar plate-reader (λ_exc = 350/15 nm, λ_em = 433/20 nm). All signals were blank (i.e. only HT-29 cells) corrected, and adhesion is expressed relative to unwashed yeast-exposed cells: (intensity adhered cells/intensity control well)*100%.

To assess the potential internalization of NFC by BEAS-2B cells, NFC was stained with the Calcofluor White Stain (Merck KGaA, Darmstadt, Germany). The staining was performed on the same slides that were used for the scoring of the micronucleus frequency (for slide preparation, see below). At harvest, the cells were treated for 15 min at 25 °C with cellulase (8.7 µL/mL, Cellic CTec2, Novozymes, Bagesvaerd, Denmark) to remove excess NFC outside the cells. The procedure of the calcofluor staining has previously been described [35 (link)]. In brief, a drop of calcofluor was applied onto acridine orange-stained slides and incubated under a cover slip for 1 min. The sample was thereafter examined with a fluorescence microscope (ZEISS Axio Imager Z1, Carl Zeiss AG, Oberkochen, Germany), using DAPI/FITC/TRITC triple filter and 40x objective lens.

The mixed
solutions containing E. coli ER2738
(4.3 × 10⁷ cfu mL^–1), surface-aminated
or plain cellulose assemblies [0.02% (w/v)], and EDTA (100 μM)
were diluted 10-fold with PBS either immediately after mixing or after
incubation for 48 h at 37 °C. Eight microliters of the diluted
solutions were placed on a glass slide, mixed with 1 μL of Calcofluor
White Stain (Merck KGaA) and 10% KOH aqueous solution, and then covered
with a cover glass. After incubation for 1 min under ambient conditions,
fluorescence microscopy images were obtained using a fluorescence
microscope (Eclipse LV100ND, Nikon, Tokyo, Japan) with excitation,
dichroic mirror, and barrier filter wavelengths of 330–380,
400, and 420 nm, respectively, at room temperature.

Seedlings were fixed in 4% (w/v) paraformaldehyde solution containing 0.05% (v/v) Triton-X100 using vacuum desiccator or centrifugation to measure the leaf size and adaxial subepidermal cell size in the first foliage leaf primordia. After fixation, the samples were washed with phosphate-buffered saline (PBS) and cleared using ClearSee (Kurihara et al., 2015 (link)) containing 1% (v/v) Calcofluor White Stain (Sigma-Aldrich, Saint Louis, MO, USA) to stain the cell wall (the final calcofluor white concentration was 10 μM). For observation of nuclei, SYBR Green I Nucleic Acid Gel Stain (Lonza, Basel, Switzerland) was added to ClearSee at a 2000-fold dilution. Leaf primordia were cut under a stereomicroscope and observed under a confocal microscope (FV3000, Olympus, Tokyo, Japan or FV10i, Olympus). Calcofluor was excited with a laser at 405 nm, emission was detected between 430 and 470 nm, and SYBR Green was excited at 488 nm and emission was detected between 490 and 540 nm. The measurements were performed using ImageJ (Schneider et al., 2012 (link); RRID: SCR_003070) or Fiji (Schindelin et al., 2012 (link); RRID: SCR_002285). Leaf and cell shape were traced manually or by using ‘Morphological Segmentation’ plugin (Legland et al., 2016 (link)), and the area was measured using the “Analyze particles” function.

Cellulose production in colony biofilms was quantified by measuring the cellulose bound to cells [27 (link)]. STm WT, ∆csgD, ∆csgA, ∆bcsA, ∆csgA: ∆bcsA were spotted on LB-NaCl Agar supplemented with 2% Calcofluor white stain (Sigma Aldrich), incubated for 3 days at 25 °C. The colony biofilms formed were retrieved, mixed with 1 mL PBS, and centrifuged at 5000 rpm for 10 min to eliminate the unbound cells and Calcofluor. Cells were resuspended in water and transferred to a 96 well plate (Tarsons). Fluorescent measurements corresponding to biofilm bound cellulose (excitation 360 nm, emission 460 nm) were made in Microplate reader (Biotek). The results were analysed using GraphPad Prism5 for Windows.

A method described in our previous study was used for immunolabelling procedures^{26 (link)}. At the onset, the collected sections mounted on microscope slides were circled with a liquid blocker (Daido Sangyo, Japan). Slides prepared for immunochemistry reactions were pre-incubated with 1% bovine serum albumin (BSA, Sigma Aldrich) in PBS for 30 min to avoid nonspecific binding of antibodies. The rat IgM primary anti-AGP mAbs JIM13, MAC207, LM2, and LM14 were diluted 1:50 in 0.1% BSA. Fluorescence-labelling was based on the use of goat anti-rat IgM conjugated with AlexaFluor 488 (Cat. No. A21212; ThermoFisher Scientific, USA) adjusted to a 1:200 dilution in 0.1% BSA. After incubation with primary and secondary antibodies, the sections were washed in PBS twice and finally enclosed in Dako Fluorescent Mounting Medium (Sigma Aldrich). For better visualization of the examined epitopes, the sections were counterstained with (0.001%, w/v) Calcofluor White Stain (Sigma Aldrich). Immunofluorescence labelling was carried out on at least several serial sections from each sample of the apple fruit and each kind of antibodies. Control reactions were carried out by incubation in PBS instead of the primary antibody.