Macrophages were prepared from adult male C57BL/6 (WT) mice (Harlan) and P2X7R KO mice [60 (link)] as described previously [61 (link)]. In brief, the peritoneal cavity was lavaged with RPMI 1640 media (Sigma). Cells were collected by centrifugation (250 x g, 5 minutes) and plated in 24-well plates at a density of 5 x 105 cells/well in RPMI 1640 media (Sigma) supplemented with 5% FBS (PAA Laboratories), 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma). Cells were cultured overnight (37 °C, 5% CO2) before non-attached cells were removed by a media change. Cells were incubated with LPS (1 μg/ml for 2 hours) before treatment with LL-37 and ATP as indicated.
Rpmi 1640 media
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, Australia, Canada, Switzerland, Austria, Israel, Japan
RPMI-1640 is a widely used cell culture medium. It is a complex formulation of amino acids, vitamins, salts, and other components necessary for the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (32 mentions)
Fetal bovine serum (fbs), by Merck Group (31 mentions)
L glutamine, by Merck Group (22 mentions)
Streptomycin, by Merck Group (22 mentions)
Penicillin, by Merck Group (19 mentions)
Penicillin streptomycin, by Merck Group (15 mentions)
L glutamine, by Thermo Fisher Scientific (15 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (14 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (10 mentions)
Streptomycin, by Thermo Fisher Scientific (10 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
Dmem media, by Merck Group (8 mentions)
Penicillin, by Thermo Fisher Scientific (8 mentions)
Fetal calf serum (fcs), by Merck Group (7 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (7 mentions)
Insulin, by Merck Group (6 mentions)
Sodium pyruvate, by Merck Group (5 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (5 mentions)
Foetal calf serum, by Thermo Fisher Scientific (5 mentions)
Mm 1s, by American Type Culture Collection (5 mentions)
314 protocols using rpmi 1640 media
1
Macrophage Isolation and Stimulation Protocol
2
Isolation and Transformation of Lymphocytes
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The buffy coat layer was removed from lithium heparinized blood samples and washed with RPMI-1640 media from Sigma-Aldrich in Egypt. The cleaned cells were resuspended in 1 mL of RPMI-1640 media containing 10% fetal calf serum (Sigma-Aldrich, Egypt). The number of viable lymphocytes per mL of RPMI was determined using a Neubauer Hemocytometer and trypan blue stain (Sigma-Aldrich; St. Louis, MO, USA). Each sample was tested three times. 10 Set up for lymphocytes were cultured with phytohemagglutinin (PHA) mitogen at a concentration of 10 µg/mL. The plates were incubated for 72 h at 37 °C in an incubator with a 5 percent CO2 tension. Finally, lymphocyte transformation was carried out at 490 nm utilizing methyl thiazolyl tetrazolium reduction techniques [27 (link)]. The supernatant of cultured lymphocytes was used to collect plasma.
3
Culture Conditions for Cancer Cell Lines
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Ovarian (IGROV1, A2780, SKOV3, BG1, OVCAR3, OVCAR4, OVCAR5, OVCAR8, OAW42, TYK-nu, KURAMUCHI, OVSAHO, CAOV3, and TOV21G), and breast (T47D and MCF7) cancer cell lines, ovarian surface epithelial cells (HOSE17.1 and HOSE6.3) and normal mammary epithelial cell line MCF10A were grown in RPMI-1640 media (Sigma-Aldrich, Buchs, Switzerland) supplemented with 10% fetal bovine serum (FBS), penicillin (100U/ml) and streptomycin (100 μg/ml) (obtained from Sigma-Aldrich). Fallopian tube secretory epithelial cells (FT237 and FT240) were grown in DMEM-Ham’s F12 medium without HEPES buffer (Sigma) supplemented with UltroserTm (PALL corporation, Life Science, Switzerland), penicillin (100U/ml) and streptomycin (100 μg/ml). Another ovarian cancer cell line, EFO27 were grown in RPMI-1640 media supplemented with 20% FBS, penicillin (100U/ml), streptomycin (100 μg/ml) and 1 mM sodium pyruvate (purchased from Sigma). The culture condition for all cell lines were 37 °C in a 95% humidified atmosphere containing 5% CO2.
4
Transient Transfection of Jurkat T Cells
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Jurkat T cells in RPMI-1640 media (Sigma-Aldrich), complemented with glutamine and 10% fetal calf serum (Life Technologies), were grown in an incubator under controlled conditions of 37°C, 5% CO2, and 100% humidity. The cells were transiently transfected using the Neon® transfection system (Life Technologies). One microgram of vector DNA per shot (3 pulses of 1325 V lasting for 10 ms) per 200,000 cells was used (see manufacturer’s instructions). Twenty-five-millimeter diameter microscope coverslips were cleaned by incubation with 2% Hellmanex (Sigma-Aldrich) overnight at 42˚C and subsequently washed with MiliQ water. Prior to use, the coverslips were coated with poly-l -lysine (Sigma-Aldrich). Twenty hours after transfection, the cells were washed with PBS, resuspended in phenol red-free RPMI-1640 media (Sigma-Aldrich), seeded on the poly-l -lysine-coated coverslips, and incubated for 5 min at 37°C under 5% CO2. After a quick PBS wash the cells were fixed using 4% paraformaldehyde in PBS at 37°C for 10 min. After removal of excess liquid, the fixation was stopped with 0.1 M NH4Cl in PBS and the cells were washed with PBS. Finally, the coverslip was placed into a ChamLide holder for imaging (Live Cell Instruments).
5
Cell Line Cultivation and Transfection Protocols
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IM-9 and HL-60 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured with RPMI-1640 media (Sigma) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin–streptomycin at 37°C under 5% CO2 FBS. Glucose starvation was accomplished by washing 3× with PBS and culture in glucose-free RPMI-1640 media (Sigma) with 10% dialyzed FBS (Gemini BioProducts).
For transfection, cells were seeded on 6-well plates and then transfected with the appropriate siRNA or plasmids using the manufacturer's protocols. Typically, cells were seeded on coverslips in the 6-well plates, and then 100 nM siRNA and 4 μl of DMRIE-C reagent (Invitrogen, Carlsbad, CA, USA) were used per coverslip. The cells were incubated for 4 h in the transfection mixture, which was then replaced with fresh culture medium. For getting the IM-9-Bcl-2, IM-9-Bcl-xL, HL-60-Bcl-2 and HL-60-Bcl-xL cells, IM-9 and HL-60 cells were stably transfected with pcDNA 3.1-Bcl-2 or Bcl-xL.
For transfection, cells were seeded on 6-well plates and then transfected with the appropriate siRNA or plasmids using the manufacturer's protocols. Typically, cells were seeded on coverslips in the 6-well plates, and then 100 nM siRNA and 4 μl of DMRIE-C reagent (Invitrogen, Carlsbad, CA, USA) were used per coverslip. The cells were incubated for 4 h in the transfection mixture, which was then replaced with fresh culture medium. For getting the IM-9-Bcl-2, IM-9-Bcl-xL, HL-60-Bcl-2 and HL-60-Bcl-xL cells, IM-9 and HL-60 cells were stably transfected with pcDNA 3.1-Bcl-2 or Bcl-xL.
6
HTLV-1 Immortalized T Cell Lines
7
Aesculetin Modulates Cellular Markers
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RPMI media 1640 was obtained from the Sigma-Aldrich Chemical (St. Louis, MO, USA), as were all other reagents unless specifically stated otherwise. Fetal bovine serum (FBS), penicillin/streptomycin, and trypsin/EDTA were purchased from the Lonza (Walkersville, MD, USA). IL-8 protein was provided by R&D systems (Minneapolis, MN, USA). Antibodies of α-smooth muscle actin (α-SMA), E-cadherin, fibronectin, vimentin, collagen I, membrane type 1-matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, ZO-1, β-catenin and CXCR2 were purchased from the Santa Cruz Biotechnology (Dallas, TX, USA). Human N-cadherin antibody was supplied by Abcam (Cambridge, UK). Human collagen IV antibody was purchased from Bioss Antibodies (Woburn, MA, USA). Mouse monoclonal β-actin antibody was obtained from Sigma-Aldrich Chemical. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Jackson Immuno-Research Laboratories (West Grove, PA, USA). Essential fatty acid free bovine serum albumin (BSA) and skim milk were supplied by Becton Dickinson Company (Sparks, MD, USA). 4′,6-Diamidino-2-phenylindole (DAPI) was obtained from Santa Cruz Biotechnology.
Aesculetin (Sigma-Aldrich Chemical) was dissolved in dimethyl sulfoxide (DMSO) for live culture with cells; a final culture concentration of DMSO was <0.5 %.
Aesculetin (Sigma-Aldrich Chemical) was dissolved in dimethyl sulfoxide (DMSO) for live culture with cells; a final culture concentration of DMSO was <0.5 %.
8
Cell Culture Protocols for Immune and Liver Cells
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The cell culture work has been performed as described by us in detail previously [23 (link)]. In brief, THP-1 monocytes, RAW macrophages or SVEC4-10 endothelial cells, and HepG2 human hepatocytes were cultured in RPMI Media 1640 (Sigma-Aldrich, Taufkirchen, Germany) or Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, Taufkirchen, Germany) or Eagle’s Minimum Essential Medium (EMEM) (Sigma-Aldrich, Taufkirchen, Germany) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% l-glutamine, and 50 pM β-mercaptoethanol (Gibco; 31350–010) for THP-1 in 5% CO2 at 37 °C, respectively. The cells were passaged just before reaching confluency. For cell detachment, THP-1 and RAW cells were incubated in PBS buffer, without calcium and magnesium, supplemented with 10 mm EDTA, for 15 min on ice and then carefully detached by cell scraping, while Svec4-10 and HepG2 could be trypsinized with Trypsin-EDTA solution (Sigma-Aldrich, Taufkirchen, Germany). For differentiation into macrophages, THP-1 monocytes were incubated with 150 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich, Taufkirchen, Germany) for 24 h followed by 12 h incubation in RPMI medium.
9
Culturing Prostate Cancer Cell Lines
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Prostate cancer cell lines LNCaP (CRL-1740) and PC-3 (CRL-1435) were acquired from the American Type Culture Collection (ATCC, Inc., Manassas, VA 20110-2209 USA) [70 ,71 ]. All cells were cultured with RPMI 1640 media (pH = 7.4) (Sigma–Aldrich, Inc., St. Louis, MI, USA, R8758) [72 ] with 10% of fetal bovine serum (FBS) and incubated under standard conditions (5% CO2, 37 °C).
10
Cell Line Establishment and Maintenance
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The MGH-HCC1 cell line (source: male) was derived from a resected metastasis from a patient in this cohort. The NCI-HCC cell line (source: male) was derived from a tumor grown from PDX material obtained from the NCI Patient-Derived Models Repository (model 248138-237-R). The Nthy-ori 3-1 cell line (source: female) was purchased (Sigma; cat. #90011609) and was part of the European Collection of Authenticated Cell Cultures. All cell lines were grown in humidity-controlled incubators at 21% O2, 5% CO2, and 37°C and routinely tested for Mycoplasma (Lonza; cat. #LT07-218; last tested February 16, 2023). Cell lines were authenticated using the ATCC short tandem repeat authentication method (ATCC; cat. #135-XV), and early passage freezer stocks were used for all experiments. MGH-HCC1 and NCI-HCC cells were routinely passaged in DMEM-11995 (Thermo Fisher; cat. #11995073) media with 10% fetal bovine serum (FBS) and 50 mg/mL uridine. Nthy-ori 3-1 and Hela cells (source: female; ATCC; cat. #CCL2) were routinely passed in RPMI 1640 media (Sigma; cat. #R8758) with 10% FBS. All experiments were conducted in DMEM-11995 media with 10% FBS and 50 mg/mL uridine unless otherwise specified.
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