The human HNSCC cell line HNO223 was purchased from CSL (Cell Line Service GmbH, Eppelheim, Germany). The HNO223-Luci and HNO223-shSOX2 cell lines had their origin previously described [18 (link)]. HNO223-shSOX9, non-target and positive control cell lines were generated at the DKFZ Proteomics Core Facility using Dharmacon™ Inducible shRNA according to the manufacturer’s instruction (Cat. No. V3SH11255-02EG6662). The HNO223-Luci and HNO223-shSOX2 cell lines were kept under selection adding 60 μg/mL of ZeocinTM (Invitrogen, Berlin, Germany) in DMEM complete medium. The HNO223-shSOX9 were treated with 1μg/mL of Doxycycline (Fischer Scientific, New Hampshire, EUA, Cat. No. ICN19895510) in DMEM complete medium. All cell lines were mycoplasma free, confirmed by the PCR-based detection using a mycoplasma detection kit (Thermo Fischer Scientific, Darmstadt, Germany). Cells were cultivated with Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Invitrogen, Germany), penicillin-streptomycin 50 μg/mL (Invitrogen, Germany) and 2 M glutamine (Invitrogen, Germany). Cells were kept in humidified and sterile conditions with 5% CO2 at 37 °C.
Mycoplasma detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Mycoplasma detection kit is a laboratory product designed to detect the presence of Mycoplasma species in cell cultures. It is a tool used to ensure the quality and integrity of cell lines in research and clinical settings.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Corning (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Rpmi 1640, by PAN Biotech (3 mentions)
Hek293, by American Type Culture Collection (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (2 mentions)
D f medium, by Thermo Fisher Scientific (2 mentions)
Cisplatin, by Selleck Chemicals (2 mentions)
Hct116, by American Type Culture Collection (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Doxycycline, by Thermo Fisher Scientific (1 mentions)
Hno223, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
33 protocols using mycoplasma detection kit
1
Generation of HNSCC Cell Lines
2
Isolation and Culture of Colon Cancer Cells
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The LoVo, SW620, HCT116, SW48, and SW480 human colon cancer cell lines were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The XhCRC cells were established as previously described 28 (link). Xenograft tumors were minced and incubated in DMEM/F12 (Thermo Fisher Scientific; Waltham, MA, USA) containing 1.5 mg/mL collagenase IV (Thermo Fisher Scientific), 20 µg/mL hyaluronidase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37 °C for 1 h. To remove red blood cells, the cells were treated with red blood cell lysis buffer (Thermo Fisher Scientific) on ice for 10 min, then washed twice with PBS. Isolated single cells were stained with EpCAM and subjected to fluorescence-activated cell sorting (FACSAria II, BD Biosciences; San Jose, CA, USA) to purify EpCAM-positive tumor cells. All cell lines were cultured in DMEM (Thermo Fisher Scientific) with 10% FBS (Thermo Fisher Scientific) and incubated at 37 °C with 5% CO2 in a cell culture incubator. Mycoplasma was routinely tested using a mycoplasma detection kit (Thermo Fisher Scientific).
3
Isolation and Characterization of Primary Mouse Hepatocytes and Human Hepatocellular Carcinoma Cells
4
Cell Culture of Cancer Cell Lines
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MDA-MB-231 breast cancer and Hela cervical cell and K562 leukemia cell were used as targets, cultured in RPMI-1640 (Pan-Biotech, Aidenbach, Bavaria, Germany) medium supplemented with 10% FBS (Sigma-Aldrich Chemie GmbH, Munich, Germany) and 1% penicillin/streptomycin (P/S) (Gibco, Schwerte, Germany) at 37 °C, 5% CO2, humidified atmosphere. All the cell lines were purchased from DSMZ (Braunschweig, Germany) and mycoplasma free, as tested by mycoplasma detection kit (Thermo Fisher Scientific, Darmstadt, Germany).
5
Culturing Human Cancer and Kidney Cells
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The human gastric cancer HGC-27 and the human embryonic kidney HEK-293T cells were purchased from Cell Cock Biotech, Guangzhou, China (catalog No. CC0402 and CC4003), which were authenticated by their short tandem repeat profiles. The mycoplasma contamination was verified by using mycoplasma detection kit (Thermo Fisher). The HGC-27 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% nonessential amino acids, 100 ng/mL streptomycin, and 100 U/mL penicillin. For drug treatment, ISO (0-1000 μM), FSK (0-20 μM), or IBMX (0-500 μM) was added into the medium, respectively. The HEK-293T cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS. The cells were cultured in a humidified chamber at 37 °C with 5% CO2.
6
Generation of Murine Macrophages and Inflammasome Activation
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BMDMs were generated by culturing the mouse bone marrow cells using DMEM complete medium in the presence of 20% vol/vol L929 conditional medium26 (link)63 (link). Immortalized murine macrophages from Nlrp3−/−, Asc−/−, Capase-1−/−, Nlrc4−/−, Cathepsin B−/− mice and their corresponding wild-type control cells were generously provided by Dr Katherine Fitzgerald and as previously described26 (link), and were routinely tested for mycoplasma contamination by using Mycoplasma Detection Kit (ThermoFisher Scientific). After pretreatment with ultrapure LPS (100 ng ml−1) for primary or immortalized BMDM, the cells were then stimulated with ATP for 30 min and VLP or pseudovirus for 18 h. Plasmids were transfected into macrophages using Lipofectamine 2000 according to the manufacturer's instructions. In the experiments using chemical inhibitors, they were added 1 h before inflammasome agonists. After inflammasome agonist stimulation, culture supernatants and cell lysates were collected for ELISA and immunoblot analysis.
7
Immortalized Murine Colon Epithelial Cells
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The murine young adult mouse colon (YAMC) epithelial cell line was derived from the colonic mucosa of a transgenic mouse generated by the introduction of a temperature-sensitive, interferon-inducible, SV40 T Ag, tsA58 (Immortomouse; Whitehead et al., 1993 (link)). We refer to this line as WT in this article. The Immortomouse colon epithelial cell line (IMCE) was derived from the progeny of a cross between the Immortomouse strain and the Apc (min/+) mouse strain (Whitehead and Joseph, 1994 (link)). We refer to these as MIN cells. Both cell lines were first obtained from the Center for Colon Cancer Research at the University of South Carolina and later from a new batch from R. H. Whitehead (Vanderbilt University Medical Center). Cell lines were validated by genotyping and PCR. Cells were maintained at the permissive temperature (33°C) in full RPMI 1640 medium (2 mM glutamine, 10% fetal bovine serum [FBS], 0.5 U/ml penicillin, 100 μg/ml streptomycin, 5 U/ml murine γ-interferon, and 1% ITS [insulin, transferrin, and selenium; Cellgro]). Cos-7 cells and SW480 cells were maintained in full DMEM medium (2 mM glutamine, 10% FBS, 0.5 U/ml penicillin, and 100 μg/ml streptomycin). Cell lines were tested frequently for mycoplasma contamination using a mycoplasma detection kit from Thermo Fisher Scientific.
8
Murine Colorectal Carcinoma Cell Lines
9
Generation and Validation of AEG-1 Knockout Cells
10
Culturing Invasive Melanoma Cell Lines
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B16-BL6 cells (a highly invasive variant of the mouse malignant melanoma B16 cell line; a kind gift from Dr. Isaiah J. Fidler of the MD Anderson Cancer Center, Houston, TX, USA) were cultured in D/F medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS in a humidified incubator. A375 cells (a human malignant melanoma cell line; ATCC, Rockville, MD, USA) were also used in this study. A375 cells were cultured with RPMI medium (Thermo Fisher Scientific) supplemented with 10% FBS. All cultures were checked for mycoplasma by using both a mycoplasma detection kit (Thermo Fisher Scientific) and Hoechst 33,342 staining at regular intervals.
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