Human lung alveolar carcinoma cell line A549 was cultured in RPMI-1640 (Sigma-Aldrich, Steinheim, Germany), supplemented with 10% heat inactivated FCS (Lonza, Verviers, Belgium), 2 mM L-glutamine, 100 U/mL potassium penicillin and 100 µg/mL streptomycin sulfate (all from Sigma-Aldrich). For every assay, cells growing to 90% confluence, were starved for 24 h in RPMI-1640 containing 1% heat inactivated FCS, 2 mM L-glutamine, 100 U/mL potassium penicillin and 100 µg/mL streptomycin sulfate in culture flasks or 96-well plates, before starting examination. Subsequently, cells were stimulated with RPMI-1640 (medium), zinc sulfate, sodium pyrithione (both from Sigma-Aldrich), S-nitrosocysteine (SNOC), recombinant human EGF (ImmunoTools, Friesoythe, Germany), ionomycin (Sigma-Aldrich) and combinations of the named stimulants, as indicated in the figure legends. To generate SNOC, L-cysteine and NaNO2 (both from Sigma-Aldrich) were mixed instantly before usage [16 (link)]. Induction of apoptosis by any of the stimulants was excluded by trypan blue and annexin staining.
Potassium penicillin
Manufactured by Merck Group
Sourced in Germany
Potassium penicillin is a type of lab equipment used in the production and purification of penicillin, a widely used antibiotic. It serves as a key component in the manufacturing process, facilitating the cultivation and isolation of the penicillin compound.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin sulfate, by Merck Group (7 mentions)
L glutamine, by Merck Group (7 mentions)
Rpmi 1640 medium, by Merck Group (3 mentions)
Zinc sulfate, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Fetal calf serum (fcs), by Bio&Sell (2 mentions)
Sodium pyrithione, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Lonza (1 mentions)
Nano2, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
L cysteine, by Merck Group (1 mentions)
Lithium heparin monovette, by Sarstedt (1 mentions)
Fcs 10, by Merck Group (1 mentions)
Trypan blue staining, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Harvard Bioscience (1 mentions)
Biocoll separating solution, by Harvard Bioscience (1 mentions)
Znso4, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
8 protocols using potassium penicillin
1
Culturing A549 Lung Cancer Cells
2
Isolation of Human PBMCs from Whole Blood
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Blood was obtained via venipuncture from healthy young volunteers with informed consent and ethics committee approval (RWTH Aachen University Hospital, document No. EK023/05). PBMCs were separated by Ficoll gradient centrifugation as described by our group [46 (link)]. In short, peripheral whole blood was diluted 1:2 with PBS (Sigma-Aldrich, Steinheim, Germany) and then put gently onto Ficoll. After centrifugation, cells in the interphase were collected and washed in PBS. In between, red blood cells were lysed with distilled water. Cells were resuspended in RPMI 1640 medium (Sigma-Aldrich, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Bio&Sell, Nuremberg, Germany), 2 mM L-glutamine, 100 U/mL potassium penicillin, and 100 ug/mL streptomycin sulfate (all from Sigma-Aldrich). Cells were adjusted to concentrations indicated in the specific assays.
3
Isolation and Cultivation of PBMCs
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After informed consent was obtained, human venous blood was collected from healthy volunteer donors and anticoagulated with sodium heparin (B. Braun, Melsungen, Germany). PBMCs were isolated from whole blood, as described previously [54 (link)], and cultivated in RPMI-1640 medium (Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS) “Low Endotoxin” (Bio and Sell, Feucht, Germany), 2 mM L-glutamine, 100 U/mL potassium penicillin and 100 U/mL streptomycin sulfate (all from Sigma-Aldrich).
4
Isolation and Culture of Human PBMCs
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After overnight fasting, 15 mL of blood was collected in 7.5 mL lithium-heparin monovettes (Sarstedt, Nürnbrecht, Germany) by venipuncture. Isolation of PBMC using Biocoll Separating Solution 1.077 g/mL (Biochrom, Berlin, Germany) was performed as described elsewhere [27 (link)]. Cells were adjusted to a final concentration of 1 × 106 cells/mL in culture medium. The culture medium consisted of RPMI 1640 containing 10% fetal calf serum (FCS, Bio&Sell, Feucht, Germany), 2 mM L-glutamine, 100 U/mL potassium penicillin and 100 U/mL streptomycin sulfate (all Sigma-Aldrich, Steinheim, Germany). Incubation steps were carried out in a humidified 5% CO2 atmosphere at 37 °C.
5
Two-Way Mixed Lymphocyte Reaction with Zinc
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Whole blood samples were obtained from fit youthful donors, for isolation of peripheral blood mononuclear cells (PBMC) using ficoll–hypaque density centrifugation (Biochrom, Berlin, Germany). These cells were obtained from interface, washed two times with PBS, and suspended in RPMI 1640 that contains 2 mM L-glutamine, FCS 10% (inactivated by heating at 56°C for 30 min), 100 U/mL streptomycin sulfate, and 100 U/mL potassium penicillin (all from Sigma-Aldrich, Darmstadt, Germany). A final concentration of 2×106 cells/mL was adjusted. To prepare two-way MLC, 2×106 PBMC/mL was obtained from two genetically different donors, then incubated with RPMI media or add-ed-on 50 μM zinc sulfate for 15 min (Sigma-Aldrich, Darmstadt, Germany), and next mixed at 1:1 ratio within pyrogen-free 24-well dishes for the specified time (VWR Scientific Products, Radnor, USA). Each incubation stage was performed in humidified 5% CO2 incubator at 37°C.27 (link)
6
Isolation and Culture of Porcine PBMCs
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Porcine PBMC were isolated from sodium-heparinized whole blood samples (BD Vacutainer, Denmark). Blood was diluted in a ratio of 1:3 with PBS (Sigma-Aldrich, Germany). After density centrifugation over Biocoll separating solution with a density of 1.077 g/mL (Biochrom, Germany), cells were collected from the interface and washed with PBS. Subsequently, erythrocytes were lysed with water and remaining PBMC were washed again with PBS. Cells were resuspended in RPMI 1640 medium (Sigma-Aldrich, Germany) supplemented with 10 % heatinactivated fetal calf serum (FCS) (PAA, Germany), 2 mM L-glutamine, 100 U/mL potassium penicillin and 100 U/mL streptomycin sulfate (all from Sigma-Aldrich, Germany). Viable cells were counted with trypan blue staining (Sigma-Aldrich, Germany) and adjusted to the concentration needed for the specific assays.
7
Zinc Modulation in Asian Sea Bass Cells
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Asian sea bass fry (SF) cells [22 (link)] were a kind gift from Prof. Dr. Sek-Man Wong and Prof. Dr. Gen Hua Yue from Temasek Life Sciences Laboratory, National University of Singapore. The culture used consisted predominantly of epithelial-like cells. They were cultured in Leibovitz-15 medium with 20% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/mL potassium penicillin and 100 U/mL streptomycin sulfate (all Sigma-Aldrich, Steinheim, Germany) at 27 °C. For subcultures and before seeding for experiments, cells were washed with phosphate-buffered saline (PBS), trypsinized, rewashed, taken up in fresh medium and diluted to the desired density. For zinc adequate (ZA) conditions, regular medium was used. For zinc supplementation, the medium was adjusted to 50 µM zinc with zinc sulfate (ZnSO4, Merck, Darmstadt, Germany) or zinc amino acid (Availa® zinc, ZnAA, Zinpro, Boxmeer, The Netherlands) by adding stock solutions (100 mM) to the medium. To investigate zinc deficiency, cells were cultured in medium, treated with CHELEX resin, where magnesium and calcium (both Merck) were re-supplemented, according to [23 (link)] but no zinc was added. Cells were cultured in ZA, ZnSO4, ZnAA or CHELEX medium for 3 days, washed with PBS, trypsinized, diluted 1:16 with fresh medium and analyzed 7 days after initial seeding.
8
Culturing Cholangiocarcinoma Cell Lines
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Cell lines and culture condition. We used three CCA cell lines. HuCC-T1 and SNU-1079 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and the Korea Cell Line Bank (Seoul, Korea), respectively, and JCK cells were a kind gift from D.H. Kim, from Cheonbuk National University. The cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 100 U/ml potassium penicillin (Sigma-Aldrich), 100 g/ml streptomycin, 2 mM glutamine (Sigma-Aldrich) and 20 mM sodium bicarbonate (Sigma-Aldrich). The cells were incubated with 5% CO 2 in 95% humidity in a 37˚C chamber. The growth medium was changed every 3 days.
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