The placenta was sourced from donors undergoing caesarean sections and processed shortly after retrieval. The AM was carefully detached from the chorion and rinsed with sterile saline solution to remove residual blood. The membrane was flattened on a nitrocellulose membrane filter (Merck Millipore), with its stromal/mesenchymal side facing down, in contact with the filter. The AM was immersed in a cocktail of antibiotics: vancomycin 50 µg/ml (Hospira), polimyxin B 100 µg/ml (Biochrom), ceftazidime 240 µg/ml (Fresenius-Kabi), lincomycin 120 µg/ml at +4 °C for 24 h in sterile conditions. Antibiotics are dissolved in RPMI 1640 Medium without phenol-red (Gibco, Life technologies). The AM was then cut into pieces 3 × 3 cm2 and immersed in 4 ml RPMI-1640 medium without phenol-red (Gibco, Life technologies), supplemented with 10 % human serum albumin and 10 % dimethyl sulfoxyde (DMSO, Cryo Sure, WAK-Chemie Medical GmbH) as cryopreservant. The patches were rolled up and inserted into cryovials, which were then filled with the cryopreservation medium/DMSO. Cryopreservation was achieved using a programmable cryogenic freezer (Planer KryoSave Integra, 750-30), which triggers a controlled cooling rate, taking about an hour an half to lower the temperature from +4 °C to −140 °C. The AM patches were stored in vapor-phase liquid nitrogen at −180 °C.
Rpmi 1640 medium without phenol red
Manufactured by Thermo Fisher Scientific
Sourced in United States
RPMI 1640 medium without phenol red is a widely used cell culture medium designed for the in vitro cultivation of a variety of cell types. It is formulated without the pH indicator phenol red, which can interfere with certain experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Anti sc spike mab, by GeneTex (2 mentions)
Metalasermetthrglyglyglnglnmetgly, by Merck Group (2 mentions)
Irdye700dx ir700, by LI COR (2 mentions)
Triad multi mode microplate reader, by Dynex (2 mentions)
Vancomycin, by Pfizer (1 mentions)
Nitrocellulose filter membrane, by Merck Group (1 mentions)
Ceftazidime, by Fresenius (1 mentions)
Dnase 1, by Roche (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Cayman Chemical (1 mentions)
Heparin natrium 25000, by Ratiopharm (1 mentions)
Ionomycin, by Cayman Chemical (1 mentions)
Klebsiella pneumoniae lipopolysaccharide lps, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Methanol, by Fujifilm (1 mentions)
4 4 azobis 4 cyanovaleric acid acva, by Merck Group (1 mentions)
Ripa buffer, by Nacalai Tesque (1 mentions)
N hydroxysuccinimide, by Fujifilm (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride wscd hcl, by Peptide Institute (1 mentions)
Live dead cell staining kit, by Dojindo Laboratories (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Santa Cruz Biotechnology (1 mentions)
22 protocols using rpmi 1640 medium without phenol red
1
Cryopreservation of Amniotic Membrane
2
Monoclonal Antibody Characterization Protocol
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The anti-SA monoclonal antibody (mAb) against the SA peptidoglycan epitope (clone Staph12-569.3, murine IgG3) was purchased from QED Bioscience (San Diego, CA, USA). Anti-CA mAb (clone MC3, murine IgG3), which recognises the putative β-1,2-mannan epitope in the cell wall mannoproteins and phospholipomannans of CA, was purchased from ISCA Diagnostics (Exeter, UK). The anti-SC spike mAb was obtained from GeneTex (GTX632604; Irvine, CA, USA). Anti-T7 phage mAb (T7·Tag antibody, murine IgG2b) directed against the 11 amino acid gene 10 leader peptide (MetAlaSerMetThrGlyGlyGlnGlnMetGly) of T7 phage was purchased from Merck KGaA (Darmstadt, Germany). Anti-human epidermal growth factor receptor 2 (HER2) mAb trastuzumab (Herceptin, humanised IgG1) was purchased from Chugai Pharmaceutical (Tokyo, Japan). IRDye700DX (IR700) was purchased from LI-COR Biosciences (Lincoln NE, USA). RPMI 1640 medium without phenol red was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
3
Immune Cell Activation and Oxidative Stress
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SYTOX™ Green Nucleic Acid Stain - 5 mM Solution in DMSO (ThermoFisher Scientific, S7020), Molecular Probes™ H2DCFDA (ThermoFisher, Scientific D399), Ionomycin (calcium salt) (Cayman Chemical, 11932), Klebsiella pneumoniae lipopolysaccharide (LPS) (Sigma-Aldrich, L4268), Phorbol 12-myristate 13-acetate (PMA) (Cayman Chemical, 10008014), RPMI 1640 Medium without phenol red (ThermoFisher Scientific, 11835063), Lymphoflot Ficoll-Diatrizoate density gradient solution (Bio Rad, 824012), DNase1 (Roche, 11284932001), Heparin-Natrium-25000-ratiopharm® (Ratiopharm).
4
Hematoporphyrin Dihydrochloride Cellular Assays
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Hematoporphyrin dihydrochloride (HP) was purchased from MedChem Express (San Diego, CA, USA). RPMI-1640 medium with L-glutamine and phenol red, methanol, and N-hydroxysuccinimide were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). RPMI 1640 medium without phenol red was purchased from Thermo Fisher Scientific (San Jose, CA, USA). RIPA buffer was purchased from Nacalai Tesque (Kyoto, Japan). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (WSCD-HCl) was purchased from Peptide Institute, Inc. (Osaka, Japan). Trypsin-ethylenediaminetetraacetic acid (EDTA), [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC), 4,4′-azobis(4-cyanovaleric acid) (ACVA), and N-(3-aminopropyl)methacrylamide hydrochloride (APMAA) were purchased from Sigma-Aldrich (St Louis, MI, USA). 2,2,6,6-Tetramethyl-4-piperidinol (4-OH-TEMP) was purchased from the Tokyo Chemical Industry (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Gibco (Waltham, MA, USA). The LIVE/DEAD Cell Staining Kit was purchased from Dojindo (Kumamoto, Japan). The Apoptosis/Necrosis Assay Kit (ab176749) was purchased from Abcam (Cambridge, UK). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
5
Culturing HEK293T and THP-1 Reporter Cells
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HEK293T wild type (ATCC CRL-11268) and THP-1 wild type (ATCC TIB-202) cells were purchased from Leibniz Institute DSMZ, Braunschweig, Germany. HEK293T cells were cultured in DMEM GlutaMAX™ (Thermo Fisher Scientific, Oberhausen, Germany) supplemented with 10% (v/v) heat-inactivated FBS (Thermo Fisher Scientific), 1% (v/v) pen-strep (10,000 U/mL, Thermo Fisher Scientific), and 10 µg/mL puromycin dihydrochlorid (Carl Roth, Karlsruhe, Germany) (reporter cells only) and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. THP-1 cells were cultured in RPMI 1640 GlutaMAX™ medium (Thermo Fisher Scientific) supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) pen-strep (10,000 U/mL), and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
During screening, CEBPD::SEAP THP-1 reporter cells were cultured in RPMI 1640 medium without phenol red (Thermo Fisher Scientific), supplemented with 10% (v/v) heat-inactivated FBS, 1% (v/v) pen-strep (10,000 U/mL), and 2 mM glutamine (Thermo Fisher Scientific) and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
The HEK293T and THP-1 reporter cells generated were tested for mycoplasma contamination using a mycoplasma detection kit (Lonza, Basel, Switzerland) after cell sorting.
During screening, CEBPD::SEAP THP-1 reporter cells were cultured in RPMI 1640 medium without phenol red (Thermo Fisher Scientific), supplemented with 10% (v/v) heat-inactivated FBS, 1% (v/v) pen-strep (10,000 U/mL), and 2 mM glutamine (Thermo Fisher Scientific) and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
The HEK293T and THP-1 reporter cells generated were tested for mycoplasma contamination using a mycoplasma detection kit (Lonza, Basel, Switzerland) after cell sorting.
6
Monoclonal Antibody Characterization Protocol
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The anti-SA monoclonal antibody (mAb) against the SA peptidoglycan epitope (clone Staph12-569.3, murine IgG3) was purchased from QED Bioscience (San Diego, CA, USA). Anti-CA mAb (clone MC3, murine IgG3), which recognises the putative β-1,2-mannan epitope in the cell wall mannoproteins and phospholipomannans of CA, was purchased from ISCA Diagnostics (Exeter, UK). The anti-SC spike mAb was obtained from GeneTex (GTX632604; Irvine, CA, USA). Anti-T7 phage mAb (T7·Tag antibody, murine IgG2b) directed against the 11 amino acid gene 10 leader peptide (MetAlaSerMetThrGlyGlyGlnGlnMetGly) of T7 phage was purchased from Merck KGaA (Darmstadt, Germany). Anti-human epidermal growth factor receptor 2 (HER2) mAb trastuzumab (Herceptin, humanised IgG1) was purchased from Chugai Pharmaceutical (Tokyo, Japan). IRDye700DX (IR700) was purchased from LI-COR Biosciences (Lincoln NE, USA). RPMI 1640 medium without phenol red was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
7
Evaluating Candidalysin's Impact on Macrophage Metabolism
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To determine the metabolic activity of Candidalysin-treated macrophages, 4 × 104 hMDMs/well were co-incubated with synthetic Candidalysin in triplicates in a 96-well plate for 5 h at 37 °C and 5% CO2 in 200 µL RPMI 1640 medium without phenol red (ThermoFisher Scientific). Subsequently, 50 µL of pre-warmed 1 mg/mL XTT and 100 µg/mL coenzyme Q0 (Sigma Aldrich) diluted in RPMI were added and samples were incubated for 2 h at 37 °C. The absorbance at 450 nm was measured with a Tecan Infinite microplate reader, with reference readings at 570 and 690 nm.
8
Macrophage Viability Assay with Polyphosphates
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Macrophages were cultured in RPMI 1640 medium without phenol red (Thermo Fisher Scientific) supplemented with 100 U/ml penicillin-streptomycin and 0.1% (w/v) BSA with S-/L-polyphosphates (50 µM) for 24 h. To evaluate cell viability, the lactate dehydrogenase (LDH) activity in supernatants was measured using a colorimetric CytoTox 96® assay (Promega) according to the manufacturer’s instructions. As positive controls, untreated resting macrophages were disrupted in lysis buffer and used along with samples of supernatants. After 30 min, the substrate reaction was stopped and formazan generation measured at a wavelength of 490 nm (Opsys MR Microplate Reader).
9
XTT Cell Viability Assay after Cisplatin Treatment
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To performed cell viability assay, the cells were reverse transfected with RNA oligonucleotides in 96-well plates and treated with cisplatin for 24 h at 48 h after the reverse transfection, exactly as described above (section: cell culture, reverse transfection, and apoptosis induction). Following the cisplatin treatment (72 h after reverse transfection), the medium in each well was aspirated and replaced with 100 µl of the mixture containing: RPMI-1640 medium without phenol red (Gibco®); XTT solution (BioShop Canada Inc; dissolved in PBS from Gibco®) at the final concentration of 200 µg/ml and PMS (phenazine methosulfate) solution (Sigma-Aldrich; dissolved in PBS from Gibco®) at the final concentration of 2 µg/ml. After the incubation at 37 °C for 3 h in the dark, the absorbance of each well was measured with ELISA plate reader (Dynex Technologies Triad Multi-Mode Microplate Reader) at 450 nm. All cell groups were compared against untransfected and cisplatin-untreated cells’ control group (considered as 100% viable). Samples were prepared in triplicate.
10
XTT Cell Viability Assay for Morin and Cisplatin
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Cell viability assay was performed using: XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt; BioShop Canada Inc.) dissolved in phosphate-buffered saline (PBS) solution (Gibco®); phenazine methosulfate (PMS) solution (Promega); and RPMI-1640 medium without phenol red (Gibco®).
For the XTT assay, cells were seeded at 6 × 103 cells/100 µl medium/0.32 cm2 growth area in 96-well plates, grown overnight, and treated with morin or cisplatin for 24 h and/or 48 h. Concentrations of drugs’ solvents were corrected in all wells (including control wells) to the constant level, corresponding to the highest used concentration of a particular solvent. Following the treatments, the medium in each well was replaced with 100 µl of the mixture of RPMI-1640 medium without phenol red, XTT solution (at the final concentration of 200 µg/ml) and PMS solution (at the final concentration of 2 µg/ml), prior to incubation at 37 °C for 3 h in the dark. The absorbance of each well was measured at 450 nm with ELISA plate reader (Dynex Technologies Triad Multi-Mode Microplate Reader). All treated cells were compared against control cells (considered as 100% viable). IC50 (half maximal inhibitory concentration) values were determined for each drug at each treatment time. Samples were prepared in triplicate.
For the XTT assay, cells were seeded at 6 × 103 cells/100 µl medium/0.32 cm2 growth area in 96-well plates, grown overnight, and treated with morin or cisplatin for 24 h and/or 48 h. Concentrations of drugs’ solvents were corrected in all wells (including control wells) to the constant level, corresponding to the highest used concentration of a particular solvent. Following the treatments, the medium in each well was replaced with 100 µl of the mixture of RPMI-1640 medium without phenol red, XTT solution (at the final concentration of 200 µg/ml) and PMS solution (at the final concentration of 2 µg/ml), prior to incubation at 37 °C for 3 h in the dark. The absorbance of each well was measured at 450 nm with ELISA plate reader (Dynex Technologies Triad Multi-Mode Microplate Reader). All treated cells were compared against control cells (considered as 100% viable). IC50 (half maximal inhibitory concentration) values were determined for each drug at each treatment time. Samples were prepared in triplicate.
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