Madin-Darby canine kidney (MDCK) cells (ATCC) were cultured in Eagle’s minimal essential medium (EMEM) (Lonza) supplemented with 10% fetal bovine serum (FBS) (Greiner), 100 U/ml penicillin (PEN) (Lonza), 100 U/ml streptomycin (STR) (Lonza), 2 mM l -glutamine (l -Glu) (Lonza), 1.5 mg/ml sodium bicarbonate (NaHCO3) (Lonza), 10 mM HEPES (Lonza) and 1× nonessential amino acids (NEAA) (Lonza). 293T cells were cultured in Dulbecco modified Eagle’s medium (DMEM) (Lonza) supplemented with 10% FBS, 100 U/ml PEN, 100 U/ml STR, 2 mM l -Glu, 1 mM NaHCO3, and 1× NEAA. Vero cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Lonza) supplemented with 10% FBS, 100 U/ml PEN, 100 mg/ml STR, and 2 mM l -Glu.
L glu
Manufactured by Lonza
Sourced in United Kingdom, Switzerland
L-Glu is a laboratory product that serves as a source of the amino acid L-glutamic acid. It can be used in various applications that require this specific amino acid.
Automatically generated - may contain errors
Lab products found in correlation
Hepes, by Lonza (3 mentions)
Non essential amino acids (neaa), by Lonza (2 mentions)
Penicillin pen, by Lonza (2 mentions)
Streptomycin str, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Lonza (2 mentions)
Pen strep, by Lonza (2 mentions)
Nahco3, by Lonza (1 mentions)
L glutamine l glu, by Lonza (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Lonza (1 mentions)
Eagle s minimal essential medium, by Lonza (1 mentions)
Dulbecco s modified eagle s medium, by Lonza (1 mentions)
Sodium bicarbonate, by Lonza (1 mentions)
Nahc03, by Lonza (1 mentions)
Trypsin, by Lonza (1 mentions)
Colo205, by Thermo Fisher Scientific (1 mentions)
Colo 320dm, by Thermo Fisher Scientific (1 mentions)
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)
6 protocols using l glu
1
Cell Culture Preparation for Research
2
Culture Conditions for 293T and MDCK Cells
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Cells were grown essentially as described in ref. (32 (link)). 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Lonza, Breda, The Netherlands) supplemented with 10% fetal bovine serum (Greiner), 1% nonessential aas (NEAAs) (Lonza), 1 mM sodium pyruvate (Gibco), 2 mM glutamine (L-glu, Lonza), 100 IU/mL penicillin (PEN) (Lonza), and 100 μg/mL streptomycin (STR) (Lonza). Madin–Darby canine kidney (MDCK) cells were cultured in Eagle’s minimal essential medium (Lonza) supplemented with 10% fetal calf serum, 1% NEAAs, 1.5 mg/mL sodium bicarbonate (Lonza), 10 mM Hepes (Lonza), 2 mM glutamine, 100 IU/mL PEN, and 100 μg/mL STR.
3
Viral Titration Assay in MDCK Cells
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Virus titrations were performed as described previously [13 (link)]. Briefly, MDCK cells were inoculated with tenfold serial dilutions of virus stocks, nose swabs, throat swabs or homogenized tissue samples. Cells were washed with PBS one hour after inoculation and cultured in infection media, consisting of EMEM supplemented with 100U/ml P, 100U/ml S, 2mM L-glu, 1.5mg/ml NaHC03, 10mM Hepes, 1X NEAA and 20μg/ml trypsin (Lonza). Three days after inoculation, supernatants of cell cultures were tested for agglutinating activity using turkey erythrocytes as an indicator of virus replication. Infectious virus titers were calculated from four replicates each of the homogenized tissue samples, nose swabs and throat swabs and from ten replicates of the virus stocks by the method of Reed and Muench [14 ].
4
Authenticated Colorectal Cancer Cell Lines Protocol
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Authenticated CRC lines were obtained from ECACC: HT29 (ECACC 91072201), Colo205 (ECACC 87061208), Colo320DM (ECACC 87061205), SW480 (ECACC 87092801), and SW620 (ECACC 87051203). Authenticated primary human colonocytes were obtained from ATCC: CCD-18Co (ATCC CRL-1459) and CCD-841 CoN (ATCC CRL-1790). All lines were regularly monitored for mycoplasma contamination every 2 months by PCR40 (link), and only mycoplasma-free cells were used for studies. All cells were used for no more than 15 passages. All cells were grown at 37 °C in HEPA-filtered humidified air in 5% CO2, in media supplemented with 10% heat-inactivated FBS (Gibco, Paisley, Scotland, UK, Cat. no. 10099-141), L-Glu (2 mM, Lonza, Burton on Trent, UK, Cat. no. 17-606E) and penicillin/streptomycin (100 U/ml, Gibco, Cat. no. DE17-602E). Colo205 and Colo320DM cells were grown in RPMI medium (Gibco, Paisley, Scotland, UK, Cat. no. 31870), HT29 were grown in McCoy’s medium (Lonza, Burton on Trent, UK, Cat. no. 12-688F), SW480 and SW620 were grown in L15 medium (Lonza, Burton on Trent, UK, Cat. no. BE12-700F), and HCT-116 (17) were grown in DMEM (Gibco, Paisley, Scotland, UK, Cat. no. 21885-025). CCD-18Co and CCD-841 primary cultures were grown in EMEM (Sigma-Aldrich, Saint Louis, MO, USA, Cat. no. M2279).
5
Generation of Intestinal Organoids from Crypts
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Organoids derived from intestinal crypts were generated essentially as described (Mosa et al, 2018) . Briefly, after mechanical removal of villi, intestinal tissue was cut into small tissue pieces
(2 -4 cm) and incubated in PBS/2mM EDTA for 30 min on ice. The material was passed through a 70 µm strainer, followed by centrifugation (140xg, 5 min, 4°C). The pellet was resupended in AD-DF+++ medium (Advanced DMEM/F12 (Gibco, #12634-010), 2 mM L-Glu (Lonza, #17-602E, 100 U/ml Pen/Strep (Lonza, #17-602E), 10 mM HEPES (Sigma, #H0887) and resupended in ice-cold matrigel. 50-µl aliquots of the matrigel suspension were transferred to pre-warmed 24-well plates, allowed to solidify, then covered with complete growth medium
(2 -4 cm) and incubated in PBS/2mM EDTA for 30 min on ice. The material was passed through a 70 µm strainer, followed by centrifugation (140xg, 5 min, 4°C). The pellet was resupended in AD-DF+++ medium (Advanced DMEM/F12 (Gibco, #12634-010), 2 mM L-Glu (Lonza, #17-602E, 100 U/ml Pen/Strep (Lonza, #17-602E), 10 mM HEPES (Sigma, #H0887) and resupended in ice-cold matrigel. 50-µl aliquots of the matrigel suspension were transferred to pre-warmed 24-well plates, allowed to solidify, then covered with complete growth medium
6
Isolation and Culture of BM-MSCs
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Bone marrow was collected from the iliac crest and transported to the laboratory within an hour. BM-MSCs were cultured according to standard methods (4, 27) . Under sterile conditions, ~5-10 ml of marrow suspended in α-MEM (Hyclone, Uppsala, Sweden) containing 1% penicillin-streptomycin (Pen/Strep) and 2 mM L-Glutamine (L-Glu; Sigma, St. Louis, MO, USA) was washed thrice in 0.1 M PBS with sequential centrifugation (at 25°C and 1000 rpm). Subsequently, the cells were seeded into tissue culture flasks containing α-MEM supplemented with Pen/Strep, L-Glu, and 10% fetal bovine serum (Lonza, Basel, Switzerland) at 37°C with humidified 5% CO 2 . The non-adherent cells were removed by replacing the medium on the second day of subculture. The cells were cultured up to passage 2 under the same conditions, with medium changes every other day. At this point, ~5.0 x10 6 BM-MSCs were separated and induced into the neurogenic lineage. The remaining BM-MSCs were cultured likewise for the subsequent booster application.
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