Treponema pallidum subsp. pallidum (Nichols strain) was propagated in, and extracted from, New Zealand White rabbits as described elsewhere (Lukehart and Marra, 2007 ), and stored in liquid nitrogen. Frozen treponemal stocks were then used for in vitro culture and sub-culture of T. pallidum in the presence of Sf1Ep (NBL-11) cottontail rabbit epithelial cells (ATCC CCL-68) [American Type Culture Collection (ATCC), Rockville, MD, United States]. Continuous axenic culture of T. pallidum in the absence of mammalian cells has not been achieved, and it is believed that the direct adherence of T. pallidum to Sf1Ep cells is required for the long term replication of T. pallidum in vitro (Edmondson et al., 2018 (link)). Dissociation of T. pallidum from Sf1Ep cells was accomplished using trypsin-free dissociation buffer [2 mL: 64% cell culture grade water (Sigma Aldrich), 10% modified EBSS (Earle’s Balanced salt solution, 10×), 1% non-essential amino acids (Thermo Fisher Scientific), 0.15% sodium bicarbonate (Sigma Aldrich), 0.728% 100 mM sodium pyruvate (Sigma Aldrich), 0.136% 0.5M EDTA (Thermo Fisher Scientific), 0.16 mg dithiothreitol (DTT) (Sigma Aldrich)] followed by a low speed centrifugation step (220 × g) to separate T. pallidum from the rabbit cells, as previously described (Edmondson et al., 2018 (link); Edmondson and Norris, 2021 (link)).
Cell culture grade water
Manufactured by Merck Group
Sourced in United Kingdom, United States
Cell culture grade water is a high-purity water specifically designed for use in cell culture applications. It is processed to remove impurities, metals, and other contaminants that could interfere with cell growth and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit pad enzyme, by Merck Group (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Rat tail collagen type 1, by BD (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Penicillin streptomycin mix, by Thermo Fisher Scientific (1 mentions)
Bronchial epithelial cell growth medium bullet kit begm, by Lonza (1 mentions)
Pge2 elisa kit, by Enzo Life Sciences (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
Nisin, by Merck Group (1 mentions)
F 12k medium, by American Type Culture Collection (1 mentions)
7 protocols using cell culture grade water
1
Propagation and Purification of Treponema pallidum
2
Investigating Inflammatory Signaling in Airway Cells
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Cell culture-grade water, phosphate buffered saline (PBS), heat-inactivated fetal bovine serum (FBS), N-acetyl cysteine (NAC) and Bradford protein assay reagents were purchased from Sigma (St. Louis, MO). Bronchial epithelial cell growth medium bullet kit (BEGM) was obtained from Lonza (Walkersville, MD). Dulbecco's Modified Eagle Medium (DMEM), penicillin/streptomycin mix and trypsin-EDTA were from Invitrogen (San Diego, CA). OptEIA™ Human IL-6 and IL-8 ELISA kits and type I rat tail collagen were purchased from BD Bioscience (San Diego, CA). Trypan blue solution and chemiluminescent substrate were from HyClone (Waltham, MA) and Thermo Fisher Scientific (Rockford, IL), respectively. Antibodies against phospho-p38, total p38, COX-2, and cell lysis buffer were obtained from Cell Signaling Technology (Beverly, MA). Monoclonal anti-heme oxygenase-1 (HO-1) Ab and PGE2 ELISA kit were from Enzo Life Sciences (Farmingdale, NY). R,S-Sulforaphane (SFN) and SB203580 were purchased from LKT Laboratories (St. Paul, MN) and EMD Millipore (Bedford, MA), respectively. RNeasy Mini Kit was from Qiagen (Valencia, CA). High Capacity cDNA Reverse Transcription Kit was purchased from Applied Biosystem (Grand Island, NY).
3
Preparation of Differentiation Media
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High glucose Dulbecco's modified eagle's medium (DMEM), dbcAMP and cell culture grade water were purchased from Sigma-Aldrich (Poole, UK) and other cell culture reagents were purchased from Fisher Scientific (Loughborough, UK). cell culture grade water was used to resuspend dbcAMP sodium salt (CAS #: 16980-89-5; LOT #: SLBJ2610V), filter-sterilised through a 0.2 μm filter and stored in aliquots at −20 °C. dbcAMP was freshly diluted (1:100) into differentiation media (DM) on each day of treatment.
4
Growth and Antimicrobial Susceptibility of S. pneumoniae
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S. pneumoniae strains (Supplementary Table 1 ) were routinely grown on tryptic soy agar (EMD Chemicals, NJ) supplemented with 3% sterile sheep blood or in liquid culture in semisynthetic casein liquid medium supplemented with 0.5% yeast extract (C+Y) (ref. 49 (link)) at 37 °C in 5% CO2 unless otherwise noted. Erythromycin was added at 1 μg ml−1 and kanamycin at 300 μg ml−1 for appropriate cultures. The antimicrobial peptides LL-37 (synthesized by St Jude Children's Research Hospital Hartwell Center), LL-37 5-TAMRA (Innovagen, cat SP-5259-1), Polymyxin B (Sigma, cat 92283), Nisin (Sigma, cat N5764) or β-defensin 3 (Anaspec, cat AS-60741) were resuspended in cell culture-grade water (Sigma) before use. Cell culture experiments utilized F12K medium (ATCC) supplemented with 10% fetal bovine serum (FBS) (ATCC). Optical density for growth curves was measured at λ 620 nm in 96 well plate format on a SpectraMax 340 microplate reader (Molecular Devices) and in culture tube format on a Turner Model 340 Spectrophotometer.
5
Recombinant ID-1 Citrullination Protocol
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Recombinant human ID‐1 protein (rhID‐1) (OriGene Technologies) was citrullinated in vitro using recombinant human peptidylarginine deiminase 4 (rhPAD4) enzyme (Cayman Chemical) or rabbit PAD enzyme (Sigma‐Aldrich) as previously described 7 . The preparation of noncitrullinated ID‐1 (noncit–ID‐1) was performed similarly, with cell culture–grade water (Sigma‐Aldrich) in place of PAD enzymes.
6
In Vitro Citrullination of ID1 Protein
7
Murine Erythroid Progenitor Assay
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Sodium meta-AsIII (NaAsO2, ≥99% purity, CAS 7784-46-5, Cat. No. S7400), Iscove’s Modified Dulbecco’s Medium, and cell-culture-grade water were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s phosphate-buffered saline without Ca+2 or Mg+2 (DPBS-) and fetal bovine serum were purchased from Atlanta Biologicals (Flowery Branch, GA, USA). L-Glutamine (200 mM) and penicillin/streptomycin (10,000 (mg/mL)/10,000 (U/mL)) were purchased from Life Technologies (Grand Island, NY, USA). Serum-free, M3436 MethocultTM methylcellulose medium containing erythropoietin for mouse erythroid progenitor cells (Cat. No. 03436) was purchased from STEMCELL Technologies (Cambridge, MA, USA). The EPO Quantikine ELISA kit for mice (Cat. No. MEP00B) was purchased from R&D Systems (Minneapolis, MN, USA). Acridine orange/propidium iodide (AO/PI) (Cat. No. CS2–0106) was purchased from Nexcelom Bioscience (Manchester, UK). Flow cytometry antibodies, rat anti-mouse PE CD71 (clone R17217; Cat. No. 113803), rat anti-mouse Alexa647 TER119 (clone TER-119; Cat. No. 116218), and the Zombie Aqua Fixable Viability Stain (Cat. No. 423102) were purchased from Biolegend (San Diego, CA, USA).
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