S2 cells were cultured in Express Five SFM (Thermo Fisher Scientific) supplemented with glutamax, at 27 °C and were provided by Akhtar’s lab (MPI-IE). HepG2 liver hepatocellular carcinoma (ATCC, HB-8065) were cultured in Eagle’s minimal essential medium (EMEM, Lonza, 06-174) supplemented with 10% fetal bovine serum (Sigma), 2 mM l -glutamine (Lonza), 1.8 mM CaCl2, 1 mM sodium pyruvate (Lonza) and penicillin–streptomycin mixture (100 units/mL, Lonza), at 37 °C at 5% CO2 in 10 cm plates, up to 70–80% confluency.
Cacl2
Manufactured by Lonza
Sourced in Denmark, United States
Calcium chloride (CaCl2) is a common chemical compound used in a variety of laboratory applications. It is a white, crystalline solid that is highly soluble in water. The primary function of CaCl2 is to serve as a source of calcium ions (Ca2+) and chloride ions (Cl-) in various experimental and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Express five sfm, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin mixture, by Lonza (2 mentions)
L glutamine, by Lonza (2 mentions)
Sodium pyruvate, by Lonza (2 mentions)
Mgcl2, by Lonza (2 mentions)
1 step ultra tmb elisa, by Thermo Fisher Scientific (1 mentions)
Maxisorb immunoplates, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp, by GE Healthcare (1 mentions)
Conotoxin gvia, by Alomone (1 mentions)
Hanks balanced salt solution (hbss), by Lonza (1 mentions)
Hepes, by Lonza (1 mentions)
Bicinchoninic acid bca assay kit, by Thermo Fisher Scientific (1 mentions)
Kgm 2, by Lonza (1 mentions)
5 protocols using cacl2
1
Cell Culture Protocols for S2 and HepG2 Lines
2
Pentraxin 3 Binding Assay for Thrombospondins
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Pentraxin 3 binding to TSP1 was performed as previously described (Deban et al, 2010 ). Briefly 96‐well plates (Nunc Maxisorb immunoplates, Roskilde, Denmark) were coated overnight with TSP1 (39–78 nM) in phosphate buffer (PBS++; contains 130 mg/l (1.2 mM) CaCl2 and 100 mg/l (1.4 mM) MgCl2; Lonza). After blocking of non‐specific sites with 0.5% dry milk in PBS++ (2 h at room temperature), plates are incubated with 100 μl of PTX3 (1.4–220 nM considering a molecular weight of 45 kDa for the PTX3 monomer) in PBS++ containing 0.05% Tween 20 (PBST). After washing, plates were first incubated with rabbit anti‐PTX3 polyclonal antibody (1:2,000) and then with anti‐rabbit‐IgG labeled with horseradish peroxidase (HRP: GE Healthcare, Pittsburgh, PA, USA). The chromogen substrate 3′,5,5′‐tetramethylbenzidine (TMB; 1 Step™ ULTRA TMB‐ELISA, Thermo Scientific, Rockford, IL, USA) was added and stopped with 2 N H2SO4 before reading absorbance at 450 nm. Binding to immobilized recombinant TSP‐2 and TSP‐4 and to the TSP1 fragments C‐term; N‐term; Ca‐1; P123‐1; E123‐1; and E123CaG‐1 all used at 50 nM, was performed following the same procedure. PTX3 N‐terminal domain (8.8–220 nM) was also tested in the same setting.
3
Culturing HepG2 and S2 Cell Lines
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HepG2 liver hepatocellular carcinoma (ATCC, HB-8065TM) were cultured in Eagle’s minimal essential medium (EMEM, Lonza, 06-174) supplemented with 10% fetal bovine serum (Sigma), 2 mM L-glutamine (Lonza), 1.8 mM CaCl2, 1 mM sodium pyruvate (Lonza) and penicillin–streptomycin mixture (100 units/mL, Lonza), at 37 °C at 5% CO2 in 10 cm plates, up to 80% confluency. S2 cells were provided by Asifa Akhtar’s lab (MPI-IE) and were cultured in Express Five SFM (Thermo Fisher Scientific) supplemented with glutamax, at 27 °C.
4
Neuronal Calcium Imaging Assay
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Fluo-4 non wash was purchased from Invitrogen. Hank's balanced salt solution (HBSS), Hepes, CaCl2 and MgCl2 from Lonza. Diazepam was from Fragon; phenytoin from Fluka; conotoxin GVIA and TTX from Alomone Labs; gabapentin from TCI Chemicals; and levetiracetam and retigabine were synthetized at UCB. All other reagents were from Sigma Aldrich.
5
Corneal Epithelial Cell Culture and Bacterial Interaction
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Human telomerase immortalized corneal epithelial cells (hTCEpi; Robertson et al., 2005 (link)) were maintained at 37°C in 5% CO2 in keratinocyte growth medium (KGM-2) containing 0.15 mM CaCl2 (Lonza, Walkersville, MD, USA). Cells were grown as monolayers or multilayers on either 96-well tissue culture plates or airlifted on collagen-coated Transwell filters in 12-well tissue culture plates, for use in cytotoxicity and traversal experiments, respectively (see methods below). To prepare lysates from hTCEpi cells for treatment of bacteria, hTCEpi cells were grown on 6-well tissue culture plates until confluent, then lysed by repeated cycles of cell freezing and thawing in phosphate buffered saline (PBS) followed by centrifugation (12,000 × g, 10 min, 4°C) to remove insoluble material. The supernatant (lysate) was assessed for protein concentration [Bicinchoninic Acid (BCA) Assay Kit, Pierce Biotechnology Inc., Thermo Scientific] and exposed to P. aeruginosa at 0.5 or 1.0 mg/mL of total protein as outlined below.
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