For the treatment group, 1.960 mL of PC patient blood was treated with 40 μL of functionalized liposomes (vehicle control or TRAIL therapy) and placed in a cone-and-plate viscometer that was pre-blocked with 1% BSA (Sigma) for 15 min. The blood was treated for 4 h at a physiological shear rate of 120 s− 1 to simulate the venous shear rate [26 (link)]. After 4 h, the treated blood aliquots were removed from the viscometer using HBSS buffer (Gibco) to wash out the blood and transferred to a new tube. The CTCs were isolated as previously described and placed in culture in a 6-multiwell plate using 3 mL of RPMI media (ATCC) overnight. After, the CTCs were collected from culture and stained with PI (BD) for 15 min. These cells were then fixed and cytospun onto microscope slides as indicated in the section CTC enumeration from PC patient blood samples.
Rpmi media
Manufactured by American Type Culture Collection
RPMI media is a cell culture medium formulated to support the growth and maintenance of a variety of cell types. It provides a balanced salt solution, amino acids, vitamins, and other essential nutrients required for cell proliferation. RPMI media is commonly used in research applications involving cell culture and tissue engineering.
Automatically generated - may contain errors
Lab products found in correlation
Hbss buffer, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rpmi 1640 media, by American Type Culture Collection (1 mentions)
A549 lung cancer cell line, by American Type Culture Collection (1 mentions)
Mycoalert assay control set, by Lonza (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti hif 1α, by BD (1 mentions)
Rabbit monoclonal anti akt, by Cell Signaling Technology (1 mentions)
Rabbit polyclonal anti flag, by Cell Signaling Technology (1 mentions)
Rabbit monoclonal anti phospho akt, by Cell Signaling Technology (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Mg132, by Enzo Life Sciences (1 mentions)
Pgipz lentiviral vector, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Merck Group (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
4 well culture slide, by BD (1 mentions)
8 protocols using rpmi media
1
Functionalized Liposomes Treatment of CTC
2
HER2+ Lung Cancer Cell Cultures
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HER2 positive A549 lung cancer cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA) and subcultured in RPMI media (ATCC) with 10% FBS. A549-Red-FLuc Bioware® Brite Cell Line was purchased from PerkinElmer and subcultured in RPMI-1640 media (ATCC) with 10% FBS. Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C.
3
Cell Line Propagation and Authentication
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Cell lines were acquired from ATCC: SW1417, CCL-238, RRID: CVCL_1717; COLO320DM, CCL-220, RRID: CVCL_0219; SW403, CCL-320, RRID:CVCL_0545; and SW948, CCL-237, RRID:CVCL_0632. COLO320DM were cultured in RPMI Media (ATCC 30-2001). SW1417, SW403, and SW948 were cultured in DMEM (GIBCO 10569-010). Cell lines were maintained in humidified 37°C incubators with 5% CO2. All media were formulated using 10% FBS and 1% penicillin/streptomycin. Assay-specific media formulations are described in their corresponding sections. All cell lines were tested for Mycoplasma contamination using the Myco Alert Assay Control Set (Lonza LT07-518) following the manufacturer's instructions and used only if results were negative. SW1417, COLO320DM, and SW403 cell lines were authenticated by Labcorp. For all experiments in this study, all cell lines were grown and expanded for a maximum of 15 passages from their thawing.
4
Cell Culture and Antibody Preparation Protocol
5
Norepinephrine Regulation of Cytokine Production in LPS-stimulated THP-1 Cells
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THP-1 cells were cultured and assayed in a supplemented RPMI media (ATCC, Manassas, VA). Cells were aliquoted into 1 × 106 cells/mL samples for experiments. Dose-response curves were determined for TNFα and IL-10 production in LPS-stimulated (100 ng/mL) THP-1 cell samples incubated with norepinephrine (10-5 to 10-10 mol/L), the α-selective agonist phenylephrine (PE) (10-6 to 10-10 mol/L) and the β-selective agonist isoproterenol (IPN) (10-5 to 10-10 mol/L). LPS-stimulated THP-1 cells were also co-incubated with a fixed concentration of norepinephrine 10-6 mol/L, which provided maximal effect in agonist experiments, and the β-selective antagonist propranolol (PROP) (10-5 to 10-10 mol/L), α2-selective antagonist yohimbine (YOH) (10-6 to 10-10 mol/L) or the α1-selective antagonist prazosin (PRAZ) (10-6 to 10-10 mol/L), for generation of dose-response curves. TNFα and IL-10 production was assessed as described above. Samples were assayed in duplicate.
6
Culturing and Modifying Human ES Cell Lines
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Human ES cell lines, SK-ES1 and TC71, were obtained and cultured as previously reported [19 (link)]. For primary culture, tissue samples from SK-ES1 primary tumors and metastases were transferred into a 10 cm cell culture plate, cut into pieces and cultured in RPMI media (ATCC, Manassas, VA) supplemented with 10% FBS. After a few days, cellular outgrowth from the tissue pieces was observed. Once cells reached confluence, they were trypsinized and propagated according to standard cell culture techniques. SK-ES1 cells stably expressing NPY shRNA (SK-ES1/NPY shRNA) or non-silencing shRNA (SK-ES1/NS shRNA) were created by transduction of SK-ES1 cells with a pGIPZ lentiviral vector (Thermo Scientific, Waltham, MA) encoding the corresponding shRNAs followed by puromycin selection.
7
Cell Culture Protocols for Rin5F, HEK293, and CHO
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Rin5F cells were purchased from ATCC (RRID:CVCL_2177 ; Cat. # CRL-2058, Manassas, VA). Cells were cultured in RPMI media (ATCC, Cat. # 30–2001) plus 10% heat-inactivated fetal bovine serum. Cells were cultured at 37°C in a 5% CO2 incubator. They tested negative for mycoplasma using a MycoAlert PLUS Detection Kit (Cat. #: LT07-701, Lonza, Switzerland) and they were authenticated using Cytochrome C Oxidase 1 (COX1) gene analysis by Laragen (Culver City, CA).
HEK293 c18 cells (hereon referred to as HEK293) were a gift from Carol Eng in the laboratory of Arnold J. Berk at UCLA, but they were originally purchased from ATCC (RRID:CVCL_6974 ). Cells were cultured in DMEM media (Cat. # 11965–092, Life Technologies) plus 10% heat-inactivated fetal bovine serum and 1% pen-strep (Life Technologies). Cells were cultured at 37°C in a 5% CO2 incubator. They tested negative for mycoplasma using a MycoAlert PLUS Detection Kit and they were authenticated using STR profiling (Laragen).
CHO cells were purchased from ATCC (RRID:CVCL_0214 ; Cat. #: CCL-61). Cells were cultured in RPMI 1640 with 11 mM glucose (Sigma) with 10% FBS, and 1% pen-strep. Cells were cultured at 37°C in a 5% CO2 incubator. They tested negative for mycoplasma using a PCR-based method and they were authenticated using mRNA analysis.
HEK293 c18 cells (hereon referred to as HEK293) were a gift from Carol Eng in the laboratory of Arnold J. Berk at UCLA, but they were originally purchased from ATCC (RRID:
CHO cells were purchased from ATCC (RRID:
8
Culturing Murine Bone Marrow Cells for AZT Exposure
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