The mouse melanoma cell line (B16 cells) was obtained from the Type Culture Collection of the Chinese Academy of Sciences and cultured in RPMI 1640 medium (cat. no. SH30809.01) supplemented with 10% fetal bovine serum (cat. no. SV30208), 1% penicillin and streptomycin (cat. no. SV30010; all from HyClone; Cytiva). Cells were cultured in a humidified normoxic chamber (Thermo Fisher Scientific, Inc.) with 5% CO2 at 37°C.
Rpmi 1640 medium
Manufactured by National Collection of Authenticated Cell Cultures
Sourced in China
RPMI-1640 medium is a commonly used cell culture medium developed at Roswell Park Memorial Institute. It is a basal medium that provides essential nutrients and growth factors required for the maintenance and growth of a variety of cell types, including mammalian and human cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by National Collection of Authenticated Cell Cultures (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Recombinant human trail, by Merck Group (2 mentions)
Phospho p38, by Cell Signaling Technology (2 mentions)
Caspase 8, by Cell Signaling Technology (2 mentions)
U0126, by AbMole (2 mentions)
Phospho erk, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Phospho jnk, by Cell Signaling Technology (2 mentions)
Caspase 9, by Cell Signaling Technology (2 mentions)
Mcl 1, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Cleaved caspase 9, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Ciap1, by Cell Signaling Technology (2 mentions)
Ciap2, by Cell Signaling Technology (2 mentions)
Goat anti mouse igg hrp abs20001, by Absin (2 mentions)
Goat anti rabbit igg hrp abs20002, by Absin (2 mentions)
Sp600125, by AbMole (2 mentions)
9 protocols using rpmi 1640 medium
1
Culturing Mouse Melanoma Cell Line
2
Cell Culture Conditions for Diverse Cell Lines
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PC-3, MCF-7, HeLa, U87, HPAEpiC, and A549 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China); PC-3, MCF-7, HeLa, U87, and A549 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin; HPAEpiC cells were cultured in RPMI 1640 Medium supplemented with 10% v/v fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were at 37°C in a humidified atmosphere of 5% CO2.
3
Orthotopic 4T1 Breast Cancer Model
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All animal experiments were performed in accordance with the guidelines of the Animal Care Committee. Maximum effort was maintained in minimizing animal distress. In total, 45 female BALB/c mice (aged 8–10 weeks) were purchased from the Laboratory Animal Center. They were housed under temperature‐controlled conditions (20–26 °C) with a regular daylight cycle. The laboratory mice were provided ad libitum access to purified water and were fed a standard diet.
4T1 murine breast cancer cells (RPMI 1640 medium) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Orthotopic tumor inoculation was performed by injecting 100 μL 4T1 cells (approximately 2 × 106 cells) into the right mammary fat pad of the mice. Tumor sizes were assessed daily using a Vernier caliper, and the volume was calculated using the following formula: 0.5 × length × (width2). Afterwards, tumor tissues were obtained from 15 4T1‐bearing donor mice. After achieving a tumor size of approximately 500 mm2, the mice were euthanized, and the tumor was removed from the surrounding tissue. Next, the collected tumors were cleaned to remove necrotic tissue. Fish‐like tumor tissues were soaked in a phosphate‐buffered solution on ice and cut into small pieces (0.5–1.0 mm in diameter). Finally, the tumor tissue fragments were drawn into a 1‐mL syringe.
4T1 murine breast cancer cells (RPMI 1640 medium) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Orthotopic tumor inoculation was performed by injecting 100 μL 4T1 cells (approximately 2 × 106 cells) into the right mammary fat pad of the mice. Tumor sizes were assessed daily using a Vernier caliper, and the volume was calculated using the following formula: 0.5 × length × (width2). Afterwards, tumor tissues were obtained from 15 4T1‐bearing donor mice. After achieving a tumor size of approximately 500 mm2, the mice were euthanized, and the tumor was removed from the surrounding tissue. Next, the collected tumors were cleaned to remove necrotic tissue. Fish‐like tumor tissues were soaked in a phosphate‐buffered solution on ice and cut into small pieces (0.5–1.0 mm in diameter). Finally, the tumor tissue fragments were drawn into a 1‐mL syringe.
4
TRAIL and 5-FU Combination Therapy
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The GES-1, AGS and HGC27 cell lines were purchased from the cell bank of the Chinese Academy of Sciences and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). The incubator conditions were 37°C and 5% CO 2 (12) . FBS and RPMI-1640 medium were purchased from Gibco (New York, USA). Recombinant human TRAIL (#T9701) and 5-FU (#F6627) were purchased from Sigma-Aldrich (Munich, Germany). The small-molecule inhibitors U0126 (#M1977), SP600125 (#M2076) and SB202190 (#M2062) were obtained from AbMole (Houston, USA). Primary antibodies against the following proteins were purchased from Cell Signaling Technology (Beverly, MA, USA): TRAIL (#3219), Caspase-3 (#9662), Caspase-8 (#9746), Caspase-9 (#9502), Cleaved Caspase-3 (#9661), Cleaved Caspase-8 (#9496), Cleaved Caspase-9 (#9505), PARP (#9532), Bid (#2002), DR4 (#42533), DR5 (#8047), c-IAP1 (#7065), c-IAP2 (#3130), Bcl-2 (#4223), Mcl-1 (#94296), Erk (#4695), JNK (#9252), p38 (#8690), phospho-Erk (#4370), phospho-JNK (#4668), phospho-p38 (#4511) and β-actin (#4970). The secondary antibodies used in this study included goat anti-mouse IgG-HRP (abs20001) and goat anti-rabbit IgG-HRP (abs20002), both of which were obtained from Absin (Shanghai, China).
5
TRAIL and 5-FU Combination Therapy
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The GES-1, AGS and HGC27 cell lines were purchased from the cell bank of the Chinese Academy of Sciences and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). The incubator conditions were 37°C and 5% CO 2 (12) . FBS and RPMI-1640 medium were purchased from Gibco (New York, USA). Recombinant human TRAIL (#T9701) and 5-FU (#F6627) were purchased from Sigma-Aldrich (Munich, Germany). The small-molecule inhibitors U0126 (#M1977), SP600125 (#M2076) and SB202190 (#M2062) were obtained from AbMole (Houston, USA). Primary antibodies against the following proteins were purchased from Cell Signaling Technology (Beverly, MA, USA): TRAIL (#3219), Caspase-3 (#9662), Caspase-8 (#9746), Caspase-9 (#9502), Cleaved Caspase-3 (#9661), Cleaved Caspase-8 (#9496), Cleaved Caspase-9 (#9505), PARP (#9532), Bid (#2002), DR4 (#42533), DR5 (#8047), c-IAP1 (#7065), c-IAP2 (#3130), Bcl-2 (#4223), Mcl-1 (#94296), Erk (#4695), JNK (#9252), p38 (#8690), phospho-Erk (#4370), phospho-JNK (#4668), phospho-p38 (#4511) and β-actin (#4970). The secondary antibodies used in this study included goat anti-mouse IgG-HRP (abs20001) and goat anti-rabbit IgG-HRP (abs20002), both of which were obtained from Absin (Shanghai, China).
6
HL-60 Cell Culture and P-Selectin Binding
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Human promyelocytic leukemia HL-60 cells (Cell Bank of Chinese Academy of Sciences, Shanghai, China), constitutively expressed PSGL-1 as a ligand for P-selectin, were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 10 mg/mL streptomycin and 100 units/mL penicillin at 37 °C in a humidified atmosphere of 5% CO2 in air. RPMI-1640 medium, FBS and BSA were purchased from Sigma Chemical Co. (St Louis, MO, USA). streptomycin, penicillin and phosphate buffer saline (PBS) were obtained from Gibco BRL (Grand Island, NY). Recombinant Human P-Selectin/CD62P Fc Chimera Protein (R&D Systems, Minneapolis, MN) is a disulfide-linked homodimer, containing the Fc moiety of human IgG and the extracellular domain of human P-selectin.
7
Culturing Splenocytes and RAW264.7 Cells
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Splenocytes were purified from 6-week-old normal C57BL/6 mice as previously described [10 (link)]. They were cultured in 6-well plates (2 × 106 per well) in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin (10,000 U/ml penicillin and 10 mg/m streptomycin).
RAW264.7 cells were purchased from the Cell Bank of Chinese Academy of Sciences. Cells were spread into 6-well plates equally (2 × 106 per well), and cultured in DMEM (Gibco) with 10% FBS and 1% penicillin/streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin).
Cells were incubated with or without fucoidan (30 μg/ml) at 37 °C with 5% CO2 for 24 h [19 (link)]. Then, cells were collected for further analysis.
RAW264.7 cells were purchased from the Cell Bank of Chinese Academy of Sciences. Cells were spread into 6-well plates equally (2 × 106 per well), and cultured in DMEM (Gibco) with 10% FBS and 1% penicillin/streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin).
Cells were incubated with or without fucoidan (30 μg/ml) at 37 °C with 5% CO2 for 24 h [19 (link)]. Then, cells were collected for further analysis.
8
LUAD Cell Line Culturing Protocol
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The human LUAD cell lines A549 and H1299 were procured from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences, and were cultured in RPMI-1640 medium (cat. no. A1049101; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; cat. no. 10100147; Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)/streptomycin (100 µg/ml) for <15 passages. The cells were authenticated by Shanghai Biowing Applied Biotechnology Co., Ltd. and were maintained under standard cell culture conditions at 37°C in a humidified incubator with 5% CO2.
9
Rat PC12 Cell Neuroprotection Model
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Rat PC12 cells (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences) were used in the present study and cultured in RPMI-1640 medium (cat. no. 11875093; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated horse serum (cat. no. 04-124-1A; Shanghai Yaoyun Biological Technology Co., Ltd.) and 5% fetal bovine serum (cat. no. 16000-044; Gibco; Thermo Fisher Scientific, Inc.). Cells were treated with 10 mM oxygenated hemoglobin (OxyHb) at 37˚C to construct the SAH cell model for 24 h, while the control cells were treated with corresponding PBS solution. BAY11-7082 (5 µmol/l; cat. no. S2913; Selleck Chemicals), an inhibitor of NLRP3(24 (link)), was dissolved in PBS and added to the culture medium at 37˚C for 48 h, while PBS was used to culture cells as control. Moreover, 3K3A-APC (FILZB1-03; ZZ Biotech LLC) was used to treat cells at the concentrations of 5, 10 and 20 ng/ml, respectively.
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