Bone marrow-derived macrophages (BMDM) were generated by flushing bone marrow cells from femurs and tibia of WT and Arhgef2-/- mice followed by depleting erythrocytes using Ammonium-Chloride-Potassium (ACK) lysis buffer (Gibco), passing cells through a 70 μM cell strainer. Resuspending cells in DMEM (Gibco) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Gibco), and 20 ng/mL M-CSF (eBioscience). COS-7 cells were purchased from Bioresource Collection and Research Center (BCRC) and cultured by DMEM supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). Cells were maintained at 37 °C, 5% CO2 for six days before experiments. Plasmid encoding GEF-H1 (pCMV6-Entry-hGEF-H1) was purchased from Origene.
Cos 7 cells
Manufactured by RIKEN BioResource Center
Sourced in Japan, United States
COS-7 cells are a line of fibroblast-like cells derived from the kidney of the African green monkey. They are commonly used in cell biology research as a model system for the expression and study of recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Lenti x 293t cells, by Takara Bio (1 mentions)
Crl 3216, by American Type Culture Collection (1 mentions)
D6046, by Merck Group (1 mentions)
Ccl 22, by American Type Culture Collection (1 mentions)
Hek293t cells, by RIKEN BioResource Center (1 mentions)
Pc12 cells, by RIKEN BioResource Center (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Fujifilm (1 mentions)
D d solubilizer, by Takara Bio (1 mentions)
B16f1 melanoma cells, by American Type Culture Collection (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Mcf10a cells, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Advanced mem, by Merck Group (1 mentions)
6 protocols using cos 7 cells
1
Generating Bone Marrow-Derived Macrophages
2
Cell Lines for HIV Research
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TZM-bl cells (ARP-812932 (link),33 (link),34 (link),35 (link),36 (link),37 (link)) were obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH (contributed by Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc.) Lenti-X 293T cells (632180, Takara Bio Inc.), TZM-bl cells, HEK293T cells (CRL-3216, ATCC) and COS7 cells (0539, Riken BRC Cell Bank) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (08468-16, Nacalai Tesque, Kyoto, Japan), while HeLa S3 cells (CCL-2.2, ATCC) were cultured in DMEM with low glucose (D6046, Sigma-Aldrich, St. Louis, MO, USA), supplied with 10% (v/v) fetal bovine serum (FBS) (173012, Sigma-Aldrich). HeLa CD4+ cells (154, NIH AIDS reagent program) were maintained in DMEM supplemented with 10% (v/v) FBS and 2 mg/mL G418 (16512-81, Nacalai Tesque, Kyoto, Japan). MT4 cells (JCRB1216, Japanese Collection of Research Bioresources Cell Bank), C8166-CCR5 cells, and THP-1 cells were cultured in RPMI-1640 (R8758, Sigma-Aldrich) supplemented with 10% (v/v) FBS. For passaging the adherent cells, cells were treated with 2.5 g/L-trypsin/EDTA solution (32777-44, Nacalai Tesque). All the cells were incubated in a humidified incubator at 37°C with 5% CO2.
3
Cell Line Source and Handling
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COS7 cells, HEK293T cells, and PC12 cells were obtained from RIKEN Bioresource Center Cell Bank (https://cell.brc.riken.jp/ ). PC12D cells were provided by Dr. Shinichi Hisanaga (Tokyo Metropolitan University) and were originally developed in Katoh-Semba et al. 64 (link). No commonly misidentified cell lines were used in this study. The cell lines were not tested for mycoplasma contamination. None of the cell lines used were authenticated.
4
B16-F1 Melanoma Cell Culture and Manipulation
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The B16-F1 melanoma cells (obtained from the American Type Culture Collection, Manassas, VA, USA), Tyr-KO B16-F1 cells21 (link), and COS-7 cells (obtained from RIKEN BioResource Research Center, Tsukuba, Japan) were cultured at 37 °C under 5% CO2 in D-MEM medium (FUJIFILM Wako Pure Chemical, Osaka, Japan) containing 10% fetal bovine serum, 100 units/mL penicillin G, and 100 µg/mL streptomycin. In the KD experiments, B16-F1 cells were transfected with siRNAs (final concentration 100 nM) by using RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, and then cultured for 48 h. In the immunofluorescence analysis, B16-F1 cells were transfected with plasmid DNAs by using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions, and then cultured for 24–72 h. In the Tyr-EGFP-FM4 synchronized transport assays, cells were treated with 500 nM D/D solubilizer (Takara Bio, Shiga, Japan) at 24 h after transfection.
5
3D Culture of MCF10A Cells
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COS-7 cells were obtained from RIKEN Bioresource Center and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). MCF10A cells were obtained from ATCC and cultured in DMEM/Ham's F-12 media supplemented with 5% horse serum, 20 ng/mL epidermal growth factor (EGF), 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin, and penicillin/streptomycin. The 3-D on-top culture of MCF10A cells was performed, according to a previously described procedure [18 (link)], except that we used φ15-mm coverslips and 24-well culture dishes, but not 8-well chamber slides. Briefly, the cells were seeded onto a solidified layer of Matrigel and cultured in DMEM/Ham's F-12 supplemented with 2% horse serum, 5 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin, and 2% Matrigel. Media were replaced every fourth day. Plasmid DNAs were transfected using jetPEI DNA transfection reagent (Polyplus transfection; Illkirch-Graffenstaden, France). siRNAs were transfected into cells using Lipofectamine RNAiMAX (Life Technologies). The siRNAs were used at a final concentration of 5 nM.
6
Cell Culture Protocols for Cos-7 and HeLa
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Cos-7 cells (RIKEN BIORESOURCE CENTER) were cultured in DMEM (Wako) supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. HeLa cells (RIKEN BIORESOURCE CENTER) cells were cultured in Advanced MEM (Sigma) supplemented with 5% FBS, 1% penicillin, and 1% streptomycin.
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