All lung cancer paraffin sections and lung cancer tissues were obtained from the First Affiliated Hospital of Dalian Medical University. The lung cancer tissues were kept in liquid nitrogen for protein extraction. Human NSCLC A549, H1299, H358 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). A549, H1299, H358 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (TransGen) and 1% penicillin-streptomycin (TransGen) at 37°C in humidified air containing 5% CO2.
Rpmi 1640 medium
Manufactured by Transgene
Sourced in China
RPMI 1640 medium is a commonly used cell culture medium formulation. It provides a balanced salt solution and essential nutrients required for the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
5 protocols using rpmi 1640 medium
1
Lung Cancer Tissue and Cell Line Protocol
2
Epidemiological Analysis of Novel PRRS Viruses
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Clinical samples, including lungs and lymph nodes, were collected from diseased piglets at two different pig farms in Sichuan province in Southwestern China during 2016–2017. The affected pigs had a high fever (40.2–42.1 °C) and exhibited overt signs of respiratory illness. Morbidity rates were 56.2%/45.3% (SCcd16/SCya17) and mortality rates were 7.3%/4.8%. To fully understand the evolutionary process of the two PRRS viruses that emerged in Southwestern China and to aid prevention and control policies against the disease, a genomic scale analysis was necessary. Clinical tissues were homogenized in an RPMI-1640 medium (Transgene, Beijing, China) using TissueLyser (Beijing, China) for RNA extraction and virus isolation. The samples that were not analyzed immediately were kept at −70 °C until use.
3
Cell Lines Culturing Conditions
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CRC cell lines (T84, LoVo, SNU-175, CL-34) and FHC (fetal human cells), purchased from the Chinese Academy of Sciences (Shanghai, China), were inoculated in RPMI 1640 medium (Transgene, Beijing, China) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) at 37°C humidified incubator with 5% CO2.
4
Culturing 786-O and A498 Cell Lines
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The 786-O cell lines (iCell-h235, Homo sapiens, CVCL number: CVCL-1051) and A498 cell lines (iCell-h235, Homo sapiens, CVCL number: CVCL-1056) were obtained and performed a Short Tandem Repeat (STR) profiling from iCell Bioscience Inc (Shanghai, China). 786-O and A498 cells were cultured in RPMI 1640 medium (FI201-01, TransGen Biotech, China) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin–streptomycin at 37 °C and 5% CO2. When the cell growth density reached 80%, the old medium was discarded, digested with 0.25% trypsin, and resuspended by adding fresh medium.
5
Isolation and Characterization of PRRSV Strain SCcd17
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The SCcd17 strain was isolated using porcine pulmonary alveolar macrophages (PAMs) from the lung tissues of diseased pigs collected from one pig farm in Sichuan Province, Southwestern China, in 2017. The pigs showed high fever and obvious respiratory signs. Lung samples were inoculated into PAMs and cultured in RPMI-1640 medium (Transgen, Beijing, China), supplemented with 5% fetal bovine serum (HyClone, South Logan, UT, USA) at 37 °C under a humid 5% CO2 atmosphere. The presence of the virus was confirmed by daily observation of cytopathic effects (CPE) and an indirect immunofluorescent assay (IFA) using anti-N protein of PRRSV monoclonal antibody, GTX (GeneTex, Irvine, CA, USA). The virus was purified twice by plaque assay and the PRRSV isolate SCwhn09CD (isolated in 2009 in Sichuan Province) was used as a positive control in the plaque assay [31 (link)]. The purified viruses were used for complete genome sequencing. The genomic sequences of PRRSV strain SCcd17 have been submitted to GenBank database and were assigned accession no. MG914067.
The collection of PAMs and virus-containing tissue samples in this study was performed in strict accordance with the guidelines for the care and use of laboratory animals approved by the Animal Ethics Committee (AEC) of Sichuan University (Chengdu, China, 15 January 2017, SYXK [Chuan] 2013-185).
The collection of PAMs and virus-containing tissue samples in this study was performed in strict accordance with the guidelines for the care and use of laboratory animals approved by the Animal Ethics Committee (AEC) of Sichuan University (Chengdu, China, 15 January 2017, SYXK [Chuan] 2013-185).
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