Frozen PBMCs were thawed, washed twice in RPMI (Invitrogen) with 10% human AB serum, and rested at 37°C overnight. Cells were suspended in RPMI with 10% human AB serum, HEPES (Gibco BRL), glutamine, penicillin, streptomycin (Irvine Scientific), and 2‐mercaptoethanol (Sigma‐Aldrich) and cultured in 24‐well plates at a concentration of 1–3 × 106 cells/mL with 50 ng/mL PMA (Sigma) and 1 mM ionomycin (Sigma). One microlitre of Brefeldin A (BD Bioscience) per millitre medium was added at the beginning of the culture. After 4 h cultured cells were washed twice. Th1/Tc1, Th2/Tc2, and Th17/Tc17 cells were identified by their intracellular co‐expression of IFN‐γ and the transcription factor T‐bet for Th1/Tc1,36 IL‐4 and GATA‐3 for Th2/Tc2,43 and IL‐17 and the transcription factor RORγt for Th17/Tc1744 (Table3 C). The frequency of cells co‐expressing cytokines and transcription factors was measured by intracellular stain and flow cytometry. In addition, the relative frequency of CD4+ or CD8+ T cells that express IL‐2, IL‐4, IFN‐γ, TNF‐α, or IL‐17 was determined by intracellular stain. All cytokine‐positive T cell subsets were analysed in blinded fashion.
Penicillin
Manufactured by Irvine Scientific
Sourced in United States
Penicillin is a laboratory product that functions as an antibiotic. It is used in cell culture applications to prevent bacterial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Irvine Scientific (12 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (6 mentions)
Hepes, by Thermo Fisher Scientific (4 mentions)
2 mercaptoethanol, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Irvine Scientific (3 mentions)
Glutamine, by Irvine Scientific (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Brefeldin a, by BD (2 mentions)
Rpmi 1640, by Irvine Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Irvine Scientific (2 mentions)
Panc 1, by American Type Culture Collection (2 mentions)
Mycoplasma stain assay kit, by Beyotime (2 mentions)
Phenol red, by Irvine Scientific (2 mentions)
L glutamine, by Irvine Scientific (2 mentions)
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Merck Group (1 mentions)
Mhtf hepes, by Irvine Scientific (1 mentions)
F 12k medium, by American Type Culture Collection (1 mentions)
Rna clean and concentrator kit, by Zymo Research (1 mentions)
13 protocols using penicillin
1
Functional T Cell Subset Analysis
2
Culturing Human Pancreatic Cancer Cell Lines
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The PANC-1 and BxPC-3 human pancreatic cancer cell lines (American Type Culture Collection, Manassas, VA, USA) were respectively cultured in DMEM and RPMI 1640 medium supplemented with fetal bovine serum (10%), penicillin (100 U/ml), and streptomycin (100 mg/ml) (Irvine Scientific, Irvine, CA, USA). All cells were maintained at 37°C in humidified air with 5% CO2. All other reagents were obtained from HyClone China Ltd., Beijing, China. Testing for mycoplasma contamination was done using a Mycoplasma Stain Assay Kit (Beyotime Institute of Biotechnology, Beijing, China). No cell cultures were contaminated with mycoplasma.
3
Isolation of Murine Reproductive Cells
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Reproductive tracts from GFP-CETN2-expressing males were collected intact into warm PBS after sacrificed by IACUC-approved protocols. After fat pad and mesenchymal tissues removal, the reproductive tracts were dissected into sections (testis, caput, upper corpus, middle upper corpus, mid-corpus, lower corpus, cauda, vas deferens) and placed individually into modified Human Tubal Fluid (HTF) medium without calcium or protein supplementation [mHTF-Hepes: 97.8 mM NaCl/4.69 mM KCl/0.20 mM MgSO4▪7H2O/0.37 mM KH2PO4/4.0 mM NaHCO3/21.0 mM HEPES/2.78 mM glucose/21.4 mM sodium lactate/100 U/ml penicillin/100 μg/ml streptomycin/5 mg/liter phenol red; formulation modified from Irvine Scientific, Santa Ana, CA] kept at 37°. Testicular cells and epididymal sperm were extruded with fine forceps or a sterile 30-guage needle, with care taken to avoid sample mixing. Testicular cells and sperm suspensions were passed through sterile 40-μm nylon mesh filters (Fisher Scientific, Pittsburgh, PA) to remove large cellular debris, centrifuged at 400 × g for 10 minutes at room temperature, and pellets suspended in fresh warm mHTF-Hepes at 37 °C until use.
4
Aspergillus fumigatus Pathogenesis in A549 Cells
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Aspergillus fumigatus strains Af293 and CEA10 were grown on Sabouraud agar (Difco) at 37 °C for 5–7 days prior to use. The conidia were harvested by rinsing the plate with PBS containing 0.1 % Tween 80 (Sigma-Aldrich) and enumerated using a haemacytometer. For use in the experiments, the A549 type II pneumocyte cell line [American Type Culture Collection (ATCC)] was grown in F-12 K medium (ATCC) containing 10 % FBS (Gemini Bio-Products) and 1 % streptomycin and penicillin (Irvine Scientific) in 5 % CO2 at 37 °C. The day before the experiments, approximately 7×105 A549 cells in 2 ml of growth medium were cultured in each well of a six-well tissue culture plate. The next day, the A549 cells were gently rinsed twice with serum-free F-12 K medium. Next, 1×106 conidia of each strain in 2 ml of F-12 K medium were added to individual wells of the six-well tissue culture plate. Control wells contained uninfected A549 cells and organisms incubated in F-12 K medium in the absence of A549 cells. After 6 and 16 h of incubation, RNA was extracted from the A549 cells and from A. fumigatus strains using the RiboPure Kit (Ambion, Life Technology) and purified using the RNA Clean and Concentrator Kit (Zymo Research).
5
Intracellular Cytokine Profiling of T-Cell Subsets
6
T Cell Cytokine Profiling Protocol
7
Culturing Human Pancreatic Cancer Cell Lines
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The human pancreatic cancer cell lines PANC-1, BxPC-3, SW1990, and CFPAC-1 were obtained from the American Type Culture Collection (Rockville, USA) and were cultured in DMEM or RPMI 1640 medium supplemented with fetal bovine serum (10%), penicillin (100 U/mL), and streptomycin (100 mg/mL) (Irvine Scientific, Irvine, CA). All cells were maintained at 37°C in humidified air with 5% CO2. All reagents were from HyClone China Ltd. (China). Mycoplasma contamination was tested using the Mycoplasma Stain Assay Kit (Beyotime Institute of Biotechnology, Beijing, China); none of the cell cultures were contaminated with mycoplasma.
8
Preparation and Labeling of HTF Medium
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Human tubal fluid (HTF) medium (5.94 g/L NaCl, 0.35 g/L KCl, 0.05 g/L MgSO4⋅7H2O, 0.05 g/L KH2 PO4, 0.3 g/L CaCl2 2H2O, 2.1 g/L NaHCO3, 0.51 g/L D-glucose, 0.036 g/L Na pyruvate, 2.39 g/L Na lactate, 0.06 g/L penicillin, 0.05 g/L streptomycin, 0.01 g/L phenol red) was purchased from Irvine Scientific (Santa Ana, CA, USA). Pisum sativum lectin (PSL) labeled with fluorescein isothiocyanate (FITC-PSL), poly-l -lysine, bovine serum albumin (BSA), and 4′,6-diamidino-2-phenylindole were acquired from Sigma-Aldrich S.A. Ni-NTA-agarose was purchased from Qiagen (Tecnolab SA, Buenos Aires, Argentina); DL-dithiothreitol and isopropyl-β-D-1-thiogalactopyranoside (IPTG) were purchased from ICN (Eurolab SA, Buenos Aires, Argentina). Anti-GST antibody was from Calbiochem (MERCK, Buenos Aires, Argentina). Horseradish peroxidase-coupled anti-rabbit and Cy3-labeled goat anti-rabbit antibodies were from Jackson ImnunoResearch (Seroimmuno Diagnostics, Inc., Tucker, GA, USA). A23187 was from Alomone Labs. Ltd., Jerusalem, Israel. Pelco (Ted Pella Inc., CA, USA) supplied all electron microscopy supplies. All other chemical reagents were of analytical grade and were purchased from ICN or Sigma-Aldrich.
9
Rat Peritoneal Macrophage Isolation and Culture
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Rat peritoneal macrophages were harvested as previously described [23 (link)]. Macrophages were collected from 6-OHDA and control rats, following sacrifice, 4 and 8 weeks after 6-OHDA or saline injection. Cells were cultured as a monolayer in RPMI 1640 medium (Cellgro, Mediatech Inc., Herndon, VA), supplemented with 10 % fetal bovine serum (Gemini Bio-Products, Calabasas, CA), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Irvine Scientific, Santa Ana, CA). Monolayers were washed 4 h after plating to remove non-adherent cells. Cultures were shown to be >98 % pure macrophages, as assessed by staining of non-specific esterase and macrophage-specific F4/80 mAb (data not shown).
10
Cell Culture Techniques for Cancer Research
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MCF7 cells were maintained in RPMI 1640
media (Sigma) supplemented with 10% fetal bovine serum (Irvine Scientific),
penicillin (50 U/mL), and streptomycin (50 μg/mL). WM115 cells
were grown in α-MEM (Sigma) supplemented with 10% fetal bovine
serum, penicillin (50 U/mL), and streptomycin (50 μg/mL). MCF7
and WM115 were passaged after they became 90% confluent using 1% Trypsin
for MCF7 cells and 1× Accutase (Sigma) for WM115 cells. All cells
were incubated at 37 °C in a humidified atmosphere with 5% CO2. N202 cells were maintained in DMEM High Glucose supplemented
withl -glutamine (2 mM), penicillin (100 U/mL), streptomycin
(100 U/mL), sodium pyruvate (1 mM) (Invitrogen, Carlsbad, CA), and
10% heat inactivated FBS (Omega Scientific, Tarzana, CA). All cells
were maintained at 37 °C in a humidified atmosphere with 5% CO2. Cell growth and morphology were monitored by phase-contrast
microscopy.
media (Sigma) supplemented with 10% fetal bovine serum (Irvine Scientific),
penicillin (50 U/mL), and streptomycin (50 μg/mL). WM115 cells
were grown in α-MEM (Sigma) supplemented with 10% fetal bovine
serum, penicillin (50 U/mL), and streptomycin (50 μg/mL). MCF7
and WM115 were passaged after they became 90% confluent using 1% Trypsin
for MCF7 cells and 1× Accutase (Sigma) for WM115 cells. All cells
were incubated at 37 °C in a humidified atmosphere with 5% CO2. N202 cells were maintained in DMEM High Glucose supplemented
with
(100 U/mL), sodium pyruvate (1 mM) (Invitrogen, Carlsbad, CA), and
10% heat inactivated FBS (Omega Scientific, Tarzana, CA). All cells
were maintained at 37 °C in a humidified atmosphere with 5% CO2. Cell growth and morphology were monitored by phase-contrast
microscopy.
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