To obtain cultured cells, human lymphoma cell line Daudi (JCRB9071) and Large T-transformed human embryonic kidney cell line HEK293T (RIKEN BRC2202) are cultured in D-MEM medium (Wako, 044–29765) supplemented with 10% FBS, GlutaMAX (Gibco, 25050–061), Pen/Strep (Gibco, 15140–122) or RPMI-1640 medium (Wako, 189–02025) supplemented with 10% FBS, respectively, in a humidified air condition containing 5% CO2 at 37°C. 10 μL of each con A-conjugated magnetic beads are mixed with mouse testicular cells (4×105 cells), Daudi (5×105 cells), or HEK293T (2×105 cells), respectively, and incubated for 30 min at RT. Cells bound to con A-conjugated beads are captured with a magnetic stand (FG-SSMAG2, FastGene), and numbers of con A beads-unbound cells in the supernatant are counted using a Scepter 2.0 automated cell counter (Merck) and 60 μL sensor (Fig 1C
Dmem medium
Manufactured by Fujifilm
Sourced in Japan
DMEM medium is a commonly used cell culture medium that provides essential nutrients for the growth and maintenance of various cell types in laboratory settings. It is a complex mixture of amino acids, vitamins, salts, and other components that support cell metabolism and proliferation. DMEM medium is designed to support the growth of a wide range of cell lines, including fibroblasts, epithelial cells, and many others.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Rpmi 1640 medium, by Fujifilm (7 mentions)
Penicillin, by Fujifilm (3 mentions)
Streptomycin, by Fujifilm (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Dmem ham s f 12 medium, by Fujifilm (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Scepter 2.0 automated cell counter, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Atplite kit, by PerkinElmer (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
27 protocols using dmem medium
1
Culturing and Isolating Cell Lines
2
Rat Fibroblast Culture and Viability Assay
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Cultured rat fibroblasts, which were established from the subcutaneous tissue under the back skin of a healthy Wistar S/T rat (seven weeks old, 200 g), were retrieved in the form of a single cell suspension with D-MEM medium (Wako Pure Chemical) containing 10% bovine serum. The cell suspension was diluted with the same medium to 1.5 × 104 cells/mL. This suspension was poured at 100 μL/well into the coated wells in the plates described above so that there were 1.0 × 104 human cells/well (n = 4) as a control. The cell suspension was also poured into a humidified incubator with 5% CO2 at 37°C.
For the cell culture, we used 24-well culture plates with wells 15 mm in diameter without a coating (Becton, Dickinson and Company Ltd., Franklin Lakes, USA). The 24 wells were divided into three groups: the SL group, WL group, and NL group. Fifty mg of sodium alginate and the calcium gluconate and/or physiological saline solutions were combined in the same proportions by weight. The type and volume of solution are summarized inTable 4 . One, three, five, and seven days after seeding, the viable cell number in each well was counted with the ATP assay using an ATP Lite Kit (Perkin Elmer, Waltham, USA). For each time point, four wells for each experimental group were examined.
For the cell culture, we used 24-well culture plates with wells 15 mm in diameter without a coating (Becton, Dickinson and Company Ltd., Franklin Lakes, USA). The 24 wells were divided into three groups: the SL group, WL group, and NL group. Fifty mg of sodium alginate and the calcium gluconate and/or physiological saline solutions were combined in the same proportions by weight. The type and volume of solution are summarized in
3
H9c2 Cell Differentiation Protocol
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H9c2 cells were purchased from the European Collection of Cell Culture (ECACC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) medium (WAKO) supplemented with 10% fetal bovine serum (FBS). The medium was changed to DMEM supplemented 1% FBS for differentiation.
4
TTN Reporter Minigene Transfection in HeLa Cells
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HeLa cells were cultured in DMEM Medium (FUJIFILM Wako Pure Chemical, Osaka, Japan) supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. The TTN reporter minigene and the expression vectors were transiently co-transfected in a 1:4 mixture into Hela cells using Lipofectamine (Thermo Fisher Scientific, Waltham, MA) in accordance with the manufacturer’s protocol. Fluorescence images of fluorescent proteins were acquired 24 h after transfection using EVOS (Thermo Fisher Scientific), and then the cells were harvested for total RNA preparation and RT-PCR analysis.
5
Cell Culture Protocols for Cancer Research
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HEK293 cells and A549 cells were obtained from the American Type Culture Collection (CRL-1573 and CCL-185, respectively; Manassas, VA, USA) and grown in DMEM medium (Wako Pure Chemicals Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (Life Technologies, CA, USA) and 100 units/mL penicillin and 100 mg/mL streptomycin (Wako) at 37°C in a 5% CO2 humidified chamber. A human NSCLC cell line, PC-3 cells, and PC9 cells were obtained from Health Research Resources Bank (JCRB no. JCRB0077) and RIKEN BRC (RCB4455), respectively, and both cell lines were grown in RPMI-1640 medium (Wako) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin (Wako) at 37°C in a 5% CO2 humidified chamber.
6
TALEN-mediated FOS gene editing
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293T cells were cultured in DMEM medium (Wako) supplemented with 10% foetal bovine serum. TALEN was used for genome editing of the human FOS locus. TALEN plasmids targeting the human FOS gene (TALEN-FOS-left and TALEN-FOS-right) were designed and purchased from Thermo Fisher Scientific. 293 T cells (4 × 105) were transfected with TALEN expression plasmids (4 µg each) and 0.4 µg of pcDNA3.1/Hygro(−) (Thermo Fisher Scientific) using Lipofectamine 3000 (Thermo Fisher Scientific). After 2 days, hygromycin B was added (0.4 mg/ml) and hygromycin-resistant colonies were picked and cultured.
7
Ovarian Cancer Cell Line Cultivation and 5-Aza-2'-Deoxycytidine Treatment
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The serous ovarian cancer cell lines JHOS‐2, JHOS‐4 and NIH‐OVCAR3 were obtained from RIKEN BioResource Center (Tsukuba, Japan). WI‐38 and SKOV3 (epithelial ovarian cancer) were obtained from ATCC (Manassas, VA, USA). Although these cell lines were not authenticated, relatively low passage number cells were obtained. JHOS‐2 and JHOS‐4 were maintained in DMEM/Ham's F‐12 medium (Wako, Osaka, Japan), NIH‐OVCAR3 and SKOV3 were maintained in RPMI‐1640 medium (Wako), and WI‐38 was maintained in DMEM medium (Wako). All cell lines were cultured in medium containing 5% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin‐streptomycin (Wako) at 37°C in a humidified incubator with 5% CO2. For 5‐aza‐2′‐deoxycytidine (DAC, Sigma‐Aldrich, St Louis, MO, USA) treatment, cells were treated with 500 nmol/L DAC for 3 days. Medium containing DAC was replaced every day. DNA and RNA were extracted on the 7th day following the treatment.
8
Mammalian Cell Toxicity Assay
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The
mammalian cells toxicity assay was performed as previously described.18 (link) The effect of the chemical compounds on mammalian
cells was assayed using the Cell Counting Kit-8 (DOJIN), to measure
the number of living cells. The MDCK and SH-SY5Y cells were seeded
in 100 mm dishes and maintained in DMEM medium (Wako) supplemented
with 10% FBS (GIBCO), 100 units/mL penicillin, 100 mg/mL streptomycin
(GIBCO), and 2 mMl -glutamine (GIBCO). The MDCK and SH-SY5Y
cells were seeded in 96-well assay plates (CORNING) with 100 μL
of total reaction volume at 5.0 × 103 and 1.5 ×
104 cells/well, respectively, and subsequently incubated
for 6 h and overnight, respectively, at 37 °C in 5% CO2. For cell starvation, the culture medium of the MDCK and SH-SY5Y
cells was replaced with fresh DMEM medium containing 0.25% FBS, and
the cells were then incubated overnight and for 2 days, respectively.
After cell starvation, the culture medium of the MDCK and SH-SY5Y
cells was replaced with fresh medium (0.25% FBS) containing one of
the chemical compounds or ampicillin (30 μM) as a negative control.
The MDCK and SH-SY5Y cells subsequently were incubated for 1 and 2
days, respectively. We added 10 μL/well of Cell Counting Kit-8
and after 3 h measured the absorbance (450 nm) of WST-8 formazan using
a micro plate reader (BioRad).
mammalian cells toxicity assay was performed as previously described.18 (link) The effect of the chemical compounds on mammalian
cells was assayed using the Cell Counting Kit-8 (DOJIN), to measure
the number of living cells. The MDCK and SH-SY5Y cells were seeded
in 100 mm dishes and maintained in DMEM medium (Wako) supplemented
with 10% FBS (GIBCO), 100 units/mL penicillin, 100 mg/mL streptomycin
(GIBCO), and 2 mM
cells were seeded in 96-well assay plates (CORNING) with 100 μL
of total reaction volume at 5.0 × 103 and 1.5 ×
104 cells/well, respectively, and subsequently incubated
for 6 h and overnight, respectively, at 37 °C in 5% CO2. For cell starvation, the culture medium of the MDCK and SH-SY5Y
cells was replaced with fresh DMEM medium containing 0.25% FBS, and
the cells were then incubated overnight and for 2 days, respectively.
After cell starvation, the culture medium of the MDCK and SH-SY5Y
cells was replaced with fresh medium (0.25% FBS) containing one of
the chemical compounds or ampicillin (30 μM) as a negative control.
The MDCK and SH-SY5Y cells subsequently were incubated for 1 and 2
days, respectively. We added 10 μL/well of Cell Counting Kit-8
and after 3 h measured the absorbance (450 nm) of WST-8 formazan using
a micro plate reader (BioRad).
9
Isolation and Characterization of Cell-Derived Extracellular Vesicles
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HepG2, HEK293T, and NIH3T3 cell lines was purified. Cells (2 × 106) were spread on a 10 cm dish and incubated with Dulbecco’s modified Eagle medium (DMEM) medium (FUJIFILM Wako) containing 10% fetal calf serum (FCS). EVs in the FCS were eliminated by 1,00,000 × g ultracentrifugation. Cell lines were cultured until they reached 90% confluence at 37°C with 5% CO2. Then, the culture medium was changed to fresh DMEM containing 2% FCS (2% FCS-DMEM) and incubated for 24 h. The collected conditioned culture media were centrifuged at 3,000 × g for 10 min and 10,000 × g for 30 min at 4°C, followed by microfiltration with Millipore 0.22 μm filters. Filtrated conditioned culture media were reacted with MagCapture, and cell line-derived EVs were eluted using the same procedure as that used for serum EVs. Measurement of collected EV solutions using the Pierce BCA Protein Assay Kit and NanoSight was performed according to the same procedure as that for serum. The particle numbers per frame used were 42, 50, or 39 particles/frame for recording of HepG2, HEK293T, and NIH3T3 cells in Raw mode, respectively.
10
HeLa and COS-7 Cell Culture Protocol
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HeLa cells (RCB0007, RIKEN BRC) or COS-7 (RCB0539) were grown in DMEM medium (Wako, 043-30085) with 10% fetal bovine serum. The cells were maintained under normal culture conditions with 5% CO2 and 37 °C of temperature and medium was changed every 48 h until reached 80% confluency. At this point the cells were gently washed with PBS (−) and trypsinization using 1 ml TrypLE Express (Gibco, 12605-101).
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