Cells (1×103) were trypsinized and suspended in 2 ml complete medium plus 0.33% agar (Invitrogen) and plated in 6-well plate on top of a bottom agar layer (0.66% complete medium agar). After two-week days incubation, colony sizes were measured with an ocular micrometer and colonies greater than 0.1 mm in diameter were counted. All experiments were performed in triplicate.
Complete medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Complete medium is a laboratory product that provides the necessary nutrients and growth factors for the cultivation of cells or microorganisms. It contains a balanced formulation of amino acids, vitamins, salts, and other essential components to support the growth and maintenance of various cell lines or microbial cultures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Matrigel, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Crystal violet, by Beyotime (2 mentions)
Transwell chamber, by Corning (2 mentions)
Toluidine blue solution, by Solarbio (1 mentions)
Alcian blue solution, by Solarbio (1 mentions)
Ckx41 32 inverted microscope, by Olympus (1 mentions)
Serum free medium, by Thermo Fisher Scientific (1 mentions)
Eclipse ts2, by Nikon (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
A2058, by Thermo Fisher Scientific (1 mentions)
A2058, by American Type Culture Collection (1 mentions)
Melanocyte medium, by ScienCell (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Pancoll separation medium, by PAN Biotech (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
38 protocols using complete medium
1
Soft Agar Colony Formation Assay
2
Soft Agar Colony Formation Assay
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Cells (1 × 103) were trypsinized and suspended in 2 ml complete medium plus 0.33% agar (Invitrogen) and plated in 6-well plate on top of a bottom agar layer (0.66% complete medium agar). After two-week days incubation, colony sizes were measured with an ocular micrometer and colonies greater than 0.1 mm in diameter were counted. All experiments were performed in triplicate.
3
Transwell-Based Migration and Invasion Assay
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The Transwell chamber with the 24-well plate (Corning Incorporated, Corning, NY, USA; an aperture of 8 μm) was used in the migration test. A total of 200 μL cells were inoculated to the apical Transwell chamber, while 600 μL of complete medium (Invitrogen, Carlsbad, CA, USA) containing 10% FBS was added to the basolateral chamber. After incubation at 37ºC with 5% CO2 for 48 h, the cells were fixated using 4% paraformaldehyde for 30 min. Subsequently, after treatment with 0.2% Triton X-100 (Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 15 min, the Transwell chamber was stained using 0.05% crystal violet for 5 min. Cell migration was also detected.
Prior to the invasion test, the filter membrane was covered with 50 μL Matrigel (Sigma-Aldrich Chemical Company, St Louis, MO, USA). Next, 200 μL cells were inoculated to the apical Transwell chamber, while 600 μL complete medium (Invitrogen, Carlsbad, CA, USA) containing 10% FBS was added to the basolateral chamber. After incubation at 37ºC with 5% CO2 for 48 h, the aforementioned procedures were repeated for cell fixation and staining. Under an inverted microscope, five visual fields were randomly selected to count the number of stained cells (XDS-800D, Shanghai Caikon Optical Instrument Co., Ltd., Shanghai, China). The migration and invasion ability of tumor cells was assessed after calculating the average number of cells.
Prior to the invasion test, the filter membrane was covered with 50 μL Matrigel (Sigma-Aldrich Chemical Company, St Louis, MO, USA). Next, 200 μL cells were inoculated to the apical Transwell chamber, while 600 μL complete medium (Invitrogen, Carlsbad, CA, USA) containing 10% FBS was added to the basolateral chamber. After incubation at 37ºC with 5% CO2 for 48 h, the aforementioned procedures were repeated for cell fixation and staining. Under an inverted microscope, five visual fields were randomly selected to count the number of stained cells (XDS-800D, Shanghai Caikon Optical Instrument Co., Ltd., Shanghai, China). The migration and invasion ability of tumor cells was assessed after calculating the average number of cells.
4
Extracellular Matrix Evaluation in NP Cells
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To evaluate extracellular matrix levels, 3 × 105 NP cells were suspended in 20 µl of complete medium (Gibco, Thermo Fisher Scientific, MA, USA) and seeded at the center of a 24-well plate. After 2 h of incubation at 37 °C, 0.5 ml of complete medium (Gibco, Thermo Fisher Scientific, MA, USA) was added. After an additional 24 h of incubation, the following treatments were performed. After 3 d, the cells were subjected to staining with toluidine blue solution (Solarbio, Beijing, China) and alcian blue solution (Solarbio, Beijing, China) for 1 h at room temperature. In this experiment, NP cells were respectively treated with IL-1β and IL-1β + mannose.
5
Dendritic Cell Exosome Antigen Loading
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DCs were generated through the culture of bone marrow cells isolated from femurs of C57BL/6 mice (six‐eight‐week‐old) in complete medium (Gibco, USA) as previously described.[87
] Cells were cultured in complete medium added with murine GM‐CSF and IL‐4 (20 ng mL−1, Pepro Tech, USA) and refreshed every two days. On day 6, the cells were harvested for further analysis. Next, the medium was supplemented with TAT47‐57‐MBP87‐99A91 or TAT47‐57‐ OVA antigen (1 mg mL−1). After 24 h, exosome‐free complete medium was used. After 24 h of incubation, the supernatants were collected for further analysis.
] Cells were cultured in complete medium added with murine GM‐CSF and IL‐4 (20 ng mL−1, Pepro Tech, USA) and refreshed every two days. On day 6, the cells were harvested for further analysis. Next, the medium was supplemented with TAT47‐57‐MBP87‐99A91 or TAT47‐57‐ OVA antigen (1 mg mL−1). After 24 h, exosome‐free complete medium was used. After 24 h of incubation, the supernatants were collected for further analysis.
6
Colony Formation Assay Protocol
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For the colony formation assay, cells were seeded in 6-well plates at a density of 500 cells/well and cultured in complete medium (Thermo Fisher Scientific, Inc.). Two weeks later, the cells were fixed and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 30 min at room temperature. The number of visible colonies (>50 cells/colony) was counted using the CKX41-32 inverted microscope (magnification, ×100; Olympus Corporation).
7
Transwell Invasion Assay of Cancer Cells
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Kyse 150 and Eca 109 cells of different groups (NC, 25 and 50 nM) were cultured in 20% culture medium, trypsinised then suspended in serum-free medium (Thermo Fisher Scientific, Inc.). Cells were plated at a density of 1x105 cells/well in the upper chamber with 20 µl Matrigel. Complete medium (600 µl; Thermo Fisher Scientific, Inc.) containing 20% FBS was added to the lower chamber. Samples were then routinely cultured for 24 h at 37˚C and washed twice with PBS. Following cell fixation with 4% polyoxymethylene at room temperature for 30 min and staining at 37˚C for 2 h with crystal violet, the number of cells in 5 random fields of view were counted using an optical light microscope (ECLIPSE Ts2; Nikon Corporation) at x200 magnification.
8
Culturing Human Cervical Cell Lines
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Human CC cell lines (SiHa, HeLa, HCC94, and SW756) and normal cervical epithelial cells (H8) were supplied by the BeNa Culture Collection (Beijing, China). SiHa cells were cultured in DMEM (HyClone, UT, USA), while HeLa and H8 cells with RPMI 1640 medium (Thermo Fisher Scientific, MA, USA). All cells were cultured at 37 °C and 5% CO2 in a complete medium (Thermo Fisher Scientific) plus 10% FBS and 1% antibiotics.
9
Culturing Human Melanocytes and Cell Lines
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Human epidermal melanocyte (HEM) was bought from ScienCell (Carlsbad, CA, USA) and cultured in Melanocyte Medium (ScienCell). Melanoma cell lines A375 and A2058 were bought from American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (fetal bovine serum (FBS); Invitrogen, Carlsbad, CA, USA) was used for culturing A375 and A2058 cells. Human umbilical vein endothelial cells (HUVECs) were bought from ATCC. Complete medium (Thermo Fisher Scientific) was used for culturing HUVECs. All the cells were cultured in a humidified atmosphere with 5% CO2 at 37°C.
10
PBMC Isolation and Cryopreservation
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Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples anticoagulated with density gradient centrifugation (Pancoll Separation Medium; PAN Biotech) and subsequently stored at −80 °C. Frozen PBMCs were thawed in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin and 1.5% HEPES buffer 1 mol l−1 (complete medium; all additives from Thermo Fisher Scientific) until further usage.
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