Chinese hamster ovary (CHO-K1 CCL-16 from ATCC, USA) cells were cultured in Ham’s F-12K Kaighn’s modification medium (ThermoFisher Scientific) supplemented with 10% fetal calf serum and containing penicillin (100 U/mL) and streptomycin (100 µg/mL). As the CHO cells were solely used for the bimane membrane-permeability test, they were not tested for mycoplasma contamination. Cells were plated in 35 mm diameter polystyrene plates (Corning, Corning, NY) and transfected with GLIC cDNA and mCherry cDNA, as a transfection control, using a JetPRIME kit (Polyplus Transfection, Illkirch, France) according to the manufacturer’s instructions. Protein expression was allowed for 48–72 hr before cell labeling. Throughout the labeling process, cells and buffers were kept at 4°C to avoid endocytosis of the fluorophore. For labeling with BBs, cells were rinsed three times (5–10 min, gentle agitation, 4°C) in labeling buffer and incubated for 1 hr with 1 mM BBs in labeling buffer. They were then rinsed two times with labeling buffer (5–10 min, gentle agitation, 4°C) and kept in conservation buffer at room temperature during the imaging process. Each observation was made at least three times on different cell batches.
Polystyrene plate
Manufactured by Corning
Sourced in United States
Polystyrene plates are a versatile laboratory equipment used for a variety of applications. They are typically flat, rectangular dishes made of polystyrene plastic material. These plates are designed to provide a stable and clean surface for various experimental procedures, such as cell culture, assays, and sample preparation.
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Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Matrigel, by BD (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Jetprime kit, by Polyplus Transfection (1 mentions)
Ldev free hesc qualified matrigel, by Corning (1 mentions)
Accutase, by STEMCELL (1 mentions)
Ligandtracer, by Ridgeview Instruments (1 mentions)
Epoch multi volume spectrophotometer system, by Agilent Technologies (1 mentions)
Ortho phenylenediamine, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Cov434 cells, by Merck Group (1 mentions)
Golgiplug, by BD (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
K sfm medium, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
20 protocols using polystyrene plate
1
Membrane Permeability Assay with BBs
2
Maintenance of Human iPSCs in E8 Medium
3
Isolation and Culture of Human LSCs and SESCs
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Human LSCs and SESCs were isolated from donors and cultured in feeder-free plates as previously described17 (link). Briefly, the limbus tissues were isolated from postmortem human eyeballs and cut into small pieces, followed by digestion with 0.2% collagenase IV (Gibco) at 37 °C for 2 h and further digestion using 0.25% trypsin–EDTA (Gibco) at 37 °C for 15 min. Then, the cells were seeded and cultured on polystyrene plates (Corning) coated with 2% Matrigel (BD Biosciences). The components of LSC medium included DMEM/F12 and DMEM (1:1) with 1% penicillin/streptomycin, 10% fetal bovine serum, 10 ng/ml EGF, 5 μg/ml insulin, 0.4 μg/ml hydrocortisone, 0.1 nM cholera toxin, and 2 nM 3,3′,5-triiodo-l -thyronine. Human epidermal tissues were obtained from eye lids, cut into small pieces, digested, and seeded on coated polystyrene plates following the above steps. SESCs were cultured using LSC medium.
4
Kinetic Analysis of Antibody Binding to Amyloid-β Protofibrils
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LigandTracer (Ridgeview Instruments AB, Vänge, Sweden) is a technique for real-time measurement of kinetic properties of protein-protein interaction on living cells or fixated proteins [26 (link)]. Polystyrene plates (Corning Inc., Corning, NY, USA) were coated with 1 μM synthetic Aβ42 protofibrils (American Peptide, Sunnyvale, CA, USA) prepared as previously described [19 (link)], and 0.2 μM mouse TfR (mTfR; in-house produced), and incubated at 4 °C for 24 h. The plates were blocked for 2 h at room temperature with 1% BSA in PBS, and the radiolabeled antibodies used in the assay were diluted in 0.1% BSA in PBS. Binding of [125I]RmAb158-scFv8D3 and [125I]RmAb158 to Aβ protofibrils was evaluated with LigandTracer using 0.3 nM and 1 nM antibody concentrations, and the binding to mTfR was evaluated using 1 nM and 3 nM antibody concentrations. Experiments were performed in triplicate. The antibody-target interactions were analyzed in TraceDrawer v.1.8.1 (Ridgeview Instruments AB) using a 1:1 kinetic fitting model with or without depletion correction.
5
ELISA for Serum and Mucosal IgG and SIgA
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Serum samples and mucosal washes were tested for the presence of specific IgG and secretory immunoglobulin A (SIgA) using enzyme linked immunosorbent assays (ELISAs). Ninety-six well polystyrene plates (Corning) were coated with 7.5 µg/ml rTgROP17 (100 µl/well) in PBS overnight at 4°C. The plates were washed with PBS containing 0.05% Tween20 (PBST) and blocked for 1 hour at 37°C in PBS containing 5% BSA and then washed with PBS. Serum samples were diluted 1∶200 in PBS, mucosal washes without dilutions were incubated at 4°C overnight in different wells of the coated 96 well plates at 100 µl/well. Next day, the wells were washed and incubated with 100 µl of HRP-labelled goat anti-mouse antibody (AbD Serotec; diluted 1∶2500 in PBS) for serum specific IgG for 1 h at 37°C. The wells were washed extensively, incubated with orthophenylene diamine (Sigma, USA) and 0.15% H2O2 for 30 min, and enzyme reactions were terminated by the addition of 50 µl of 1 M H2SO4. Optical density (OD) was measured in an ELISA plate reader (Epoch Multi-Volume Spectrophotometer System, Biotek, USA) at 492 nm. All samples were analysed in triplicate for at least three independent experiments.
6
Culturing Hormonally Responsive COV434 Cells
7
Isolation and stimulation of intestinal immune cells
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Small intestine and colon were harvested, and mesenteric adipose tissue and Peyer’s patches removed by dissection. The intestines were opened longitudinally, washed with ice-cold HBSS-5% FBS to remove the luminal content, and cut into smaller pieces. Intestinal epithelial cells were first isolated by washing and vortexing the tissues in ice-cold PBS + 5 mM EDTA and collected for gene expression analysis. IELs were isolated using a 40–70% percoll gradient from tissue washes with ice-cold PBS + 5 mM EDTA. Intestinal lamina propria immune cells were then isolated by digestion with 1 mg/mL collagenase IV (Sigma) in complete RPMI for 40 min, at 37 °C, under agitation. Cells were washed and counted using a Neubauer’s chamber, followed by ex vivo stimulation with complete RPMI with IL-1β (10 ng/mL), IL-23 (10 ng/mL) and brefeldin A (1:1000, BD GolgiPlug), for 3 h, in 96 round-bottomed wells polystyrene plates (Corning). After this, cells were labeled with antibodies for FACS analysis (described below).
8
Biofilm Formation Assay Protocol
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Biofilm formation assays were performed as previously described with minor modifications [32 (link)]. Strains were initially grown in LB overnight at 37 °C before diluting 1 : 100 in LB, or LB supplemented with 1 % glucose, 1 % sucrose, or 1 % glycerol, and aliquoting 100 µl into 96-well, flat bottom, non-tissue culture treated polystyrene plates (Corning). Wells containing media alone were used as negative controls. Following incubation for 24 h at 37 °C, planktonic cells were removed and the wells were washed twice with dH2O. Biofilms were stained with 150 µl 0.1 % (w/v) crystal violet for 15 min and wells were rinsed twice with dH2O. Stained biofilms were solubilised with 95 % ethanol and quantified by measuring the OD600 using an Infinite M200 plate reader.
9
Limbal Stem Cell Isolation and In Vitro Differentiation
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After digestion with 0.2% collagenase IV (Gibco, # 17104019) at 37 °C for 2 h and 0.25% trypsin-EDTA (Gibco, #25200056) for another 15 min, the donors’ limbal ring biopsies were cut into small pieces and cultured on polystyrene plates (Corning) precoated with Matrigel (BD Biosciences, #354230). The components of LSC medium15 (link),42 (link) included DMEM/F12 and DMEM (1:1) with 1% penicillin/streptomycin (Gibco, #15140122), 10% fetal bovine serum (Gibco, #16000-044), 10 ng/ml EGF (millipore, #GF144), 5 μg/ml insulin (sigma, #I5500), 0.4 μg/ml hydrocortisone (Millipore, #386698), 0.1 nM cholera toxin (sigma, #C8052), and 2 nM 3,3’,5-triiodo-L-thyronine (sigma, #T2877). The medium was replaced with a fresh one every day. In order to induce in vitro differentiation of LSCs, after LSC confluence, the medium was switched to the defined K-SFM medium (Gibco, #10744-019) with 1.2 μM calcium for one week.
10
Antimicrobial Peptide Susceptibility Assay
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Equal volumes (50 μL) of bacterial suspension (2 × 106 CFU/mL) and the peptide solution were mixed in wells on polystyrene plates (Corning, USA). After the plates were incubated anaerobically for 24 h, the OD600 was measured. In negative control group, the peptides were replaced with PBS only, and chlorhexidine (CHX) was used as a positive control. The minimum inhibitory concentration (MIC) is defined as the minimum concentration that inhibits the visible growth of bacteria within 24 h. The minimum bactericidal concentration (MBC) is the minimum concentration that kills 99% of bacterial cells determined by colony counting (Cao et al., 2020 (link)).
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