Sodium cacodylate buffer

LC-MS grade water (catalog no. W6–212) and chloroform (catalog no. C6704–4) were purchased from Fisher Scientific (Hampton, NH). Carboxymethylcellulose (CMC; catalog no. C4888) was purchased from Sigma-Aldrich (St. Louis, MO). Lipid standard D-lactosyl-ß-1,1′ N-palmitoyl-D-erythro-sphingosine (LacCer(d34:1), catalog no. 860576 P) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). SEM reagents glutaraldehyde (catalog no. 16020), paraformaldehyde (catalog no. 15710), sodium cacodylate buffer (catalog no. 11652), anhydrous ethanol (catalog no. 15055), and osmium tetroxide (catalog no. 19170) were all purchased from Electron Microscopy Sciences (Hatfield, PA). Conductive silver paint (catalog no.16062) was purchased from Ted Pella, Inc. (Redding, CA).

LGG was isolated from a purchased Culturelle® LGG probiotic pill (i-Health, Inc., USA). ECN was isolated from a purchased Mutaflor® pill (Pharma-Zentrale GmbH, Germany). BL ATCC 15707 was purchased from ATCC, USA. De Man, Rogosa and Sharpe (MRS) and Tryptic soy (TS) media were purchased from Thermo Fisher Scientific, USA. Luria-Bertani (LB) media and Bacto agar were purchased from BD, USA. Carbonated sodas and beers, including Coca-Cola Classic (The Coca-Cola Company, USA), 7-Up (Keurig Dr Pepper, USA), Tiger Asian Lager (Heineken Asia Pacific, Singapore), and Guinness Foreign extra stout (Diageo, UK) were purchased from local supermarkets (see S1 Table in S2 File for their ingredient lists). Protanal CR8133 sodium alginate was purchased from FMC BioPolymer, USA. Glutaraldehyde and sodium cacodylate buffer were purchased from Electron Microscopy Services, USA. All other chemicals used in this experiment were purchased from Sigma Aldrich, USA. Sterilization by autoclaving at 121°C for 15 min was done for every media, agars, chemical, or apparatus prior to use, where necessary.

Candida alibicans (ATCC 10231) was incubated with 20µg/mL of B4010 (MIC) at 370C and 200 µl of a fungal suspension was removed after 30 minutes. The suspension was centrifuged at 2000 rpm for 2 minutes; the pellet was washed twice with phosphate buffer, prefixed in 0.5 ml of a mixed aldehyde fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer) for 24 hours. Then the pellet was washed once again in sodium cacodylate buffer (Electron Microscopy Sciences, Washington, USA) and the fungal cells suspension was subsequently mounted on poly-L-Lysine coated cover-slips and post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences). Following dehydration in a graded series of ethanol, the samples were critical-point-dried and sputter coated with 10 nm of gold. All samples were viewed and photographed on a FE SEM (Supra 55 VP - Zeiss) with an accelerating voltage of 3 kv at Carl Zeiss facility, National University of Singapore. Candida incubated in PBS was considered as positive control and processed in the same way.

PMs or PMA-differentiated macrophage-like cells in RPMI 1640 medium without antibiotics were infected with GBS or E. coli cells at a multiplicity of infection (MOI) of 20:1 unless otherwise noted. Cocultured cells were incubated at 37°C in air supplemented with 5% carbon dioxide for 1 h. As stated, some cells were pretreated with 10 μg/ml cytochalasin D (ThermoFisher), 10 nM nocodazole, 100 U/ml DNase I, 500 nM PMA, or 10 μM diphenyleneiodonium chloride (all from Sigma-Aldrich) for at least 20 min prior to infection. At 1 h, supernatants were collected, and cells were fixed with 2.0% paraformaldehyde and 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA) for at least 12 h prior to processing for microscopy.

Two- to four-week-old pilidia of M. alaskensis were relaxed for 5 to 10 minutes in a 1:1 mixture of FSW and 0.34 M MgCl₂, then killed by adding a drop of 1% formalin, and fixed by first replacing this mixture with a volume of 2.5% glutaraldehyde in 0.2 M Millonig’s phosphate buffer pH 7.4 or 0.2 M sodium cacodylate buffer pH 7.4 (Electron Microscopy Sciences, Hatfield, PA, USA), and after a few minutes, adding an equal volume of 4% OsO₄ (Electron Microscopy Sciences, Hatfield, PA, USA) for a final concentration of 1.25% glutaraldehyde and 2% OsO₄. Larvae fixed for one to two hours were rinsed in several changes of deionized water, dehydrated through an ethanol series (30% to 50% to 70%), and stored in 70% ethanol until further processing. Larvae were further dehydrated to 100% ethanol (through a series with 10% concentration increment), dried using a CO₂ EMS K850 Critical Point Drier, then sputter-coated with gold using an Emscope SC500, and examined using a Tescan Vega II scanning electron microscope.