AD293 cells (Agilent) were maintained in Dulbecco’s Modified Eagles medium (DMEM; D6546, Sigma) supplemented with 10% fetal calf serum (FCS), 1% l -glutamine, and 1% penicillin/streptomycin. CHO-K1 cells stably expressing the human SNAP_GLP1R (Cisbio) (CHO-K1-SNAP_GLP1R) were maintained in DMEM supplemented with 10% FCS, 1% penicillin/streptomycin, 500 μg/mL G418, 25 mM HEPES and 1% nonessential amino acids and 2% l -glutamine. MIN6 β-cells (a kind gift from Prof. Jun-ichi Miyazaki, Osaka University) were maintained in DMEM (D6546, Sigma) supplemented with 15% FCS, 25 mM d -glucose, 71 μM BME, 2 mM l -glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 mM HEPES. INS1 832/3 CRISPR-deleted for the endogenous GLP1R locus (a kind gift from Dr. Jacqui Naylor, MedImmune)54 (link) were transfected with human SNAP_GLP1R, before FACS of the SNAP-Surface488-positive population and selection using G41820 . The resulting SNAP_GLP1R_INS1GLP1R−/− cells were maintained in RPMI-1640 supplemented with 10% FBS, 10 mM HEPES, 2 mM l -glutamine, 1 mM pyruvate, 72 µM β-mercaptoethanol, 1% penicillin/streptomycin, and 500 μg/mL G418.
D6546
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
D6546 is a laboratory instrument designed for various analytical applications. It is capable of performing precise measurements and data analysis. The core function of D6546 is to provide accurate and reliable results for the user's research or testing needs.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (13 mentions)
L glutamine, by Merck Group (13 mentions)
Fetal bovine serum (fbs), by Merck Group (6 mentions)
F7524, by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Merck Group (5 mentions)
M7145, by Merck Group (4 mentions)
Bafilomycin a1, by Merck Group (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Merck Group (4 mentions)
P4333, by Merck Group (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
S1820 500, by Biowest (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
G7513, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Torin1, by Bio-Techne (2 mentions)
Dmem f12 medium, by Merck Group (2 mentions)
P0781, by Merck Group (2 mentions)
24 well plate, by BD (2 mentions)
55 protocols using d6546
1
Cell Culture Protocols for SNAP-GLP1R Expressing Lines
2
Isolation of Intestinal Cell Cultures
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Tph1-CFP mice were euthanized at 5–7 weeks, and a 10-cm length of small bowel or colon was removed. Full thickness tissue was inverted, chopped, and washed three times in ice-cold PBS. The tissue was digested under agitation at 37 °C in DMEM (D6546, Sigma, St. Louis, MO), 0.1% BSA (A2153, Sigma, St. Louis, Missouri), and 0.1 mg/mL (small bowel) or 0.6 mg/mL (colon) Collagenase type XI (C9407, Sigma, St. Louis, MO) in 4 separate digestions, for a total of 30 (small bowel) or 40 (colon) minutes. Supernatants were collected from the last two digestions, spun twice at 100 rcf for 5 minutes, and suspended at 1,000,000 cells per mL in DMEM (D6546, Sigma, St. Louis, MO), 5% Heat Inactivated FBS (F4135, Sigma, St. Louis, MO), 1% Pen-step (15140122, Invitrogen, Carlsbad, CA), 1% L-Glutamine (2503008, Invitrogen, Carlsbad, CA), and 10 µM (small bowel) Y-27632 (72304, Stem Cell Technologies, Vancouver, Canada) in dishes (P35GC-1.5-14-C, MatTek Corporation, Ashland, MA) coated with 5% w/v Matrigel (354230, Corning, Corning, NY). Cells were maintained in standard culture conditions for 24–48 hours.
3
Caco-2 Cell Culture for SGLT1 Expression
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The small intestinal enterocytic cell line Caco-2/TC7 (Caco-2) cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagles' medium (D6546, Sigma), supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) non-essential amino acid solution (M7145, Sigma), L-glutamine (2 mM), and penicillin/streptomycin (100 U/ml; 100 μg/ml) (basal medium). Six well plates were seeded with Caco-2 cells at 100,000 cells/cm2 and grown to confluency (~7 d) before being incubated in media containing either 0.5 mM 8-Br-cAMP, as described before (5 (link)) or a range of concentrations of VIP, PACAP, CRH, CGRP, or Substance P. Control wells were treated with basal medium only. Equal amounts of solvents, acetic acid or acetic acid with 1% (w/v) bovine serum albumin, were included in corresponding control media. The time course of the response was determined to establish that 24 h incubation period was the shortest period to give a reliable and significant difference in SGLT1 expression (data not shown). Cells were harvested after 24 h. Cell pellets were collected after centrifugation and frozen at −80°C until used.
4
Culturing Human Cell Lines for Research
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The human colon adenocarcinoma cell line Caco-2 (86010202, Sigma, St. Louis, MO, United States) was cultured in Eagle’s minimum essential medium (EMEM; 30-2003, ATCC, Manassas, VI, United States)) supplemented with 10% fetal bovine serum (FBS; 16140-071, Gibco, Gaithersburg, MD, United States), 1% non-essential amino acids (NEAA; 11140-050, Life Tech, Carlsbad, CA, United States), and 1% penicillin/streptomycin (P4333, Sigma). The human colon cell line HT29-MTX-E16 (86010202, Sigma) was cultured in Dulbecco’s modified Eagle’s medium (DMEM; D6546, Sigma) supplemented with 10% FBS (16140-071, Gibco), 1% NEAA (11140-050, Life Tech), 1% Glutamax (35050-061, Gibco), and 1% penicillin/streptomycin (P4333, Sigma). The human acute monocytic leukemia cell line THP-1 (Tib-202, ATCC) was cultured in Roswell Park Memorial Institute (RPMI) 1640 (R0883, Sigma) supplemented with 10% FBS (16140-071, Gibco), 1% Glutamax (35050-061, Gibco), and 1% penicillin/streptomycin (P4333, Sigma). The cells were tested for mycoplasma contamination and found negative.
5
Culturing and Manipulating Mammalian Cell Lines
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HeLa cells, HEK293 cells, and MEFs were cultured in DMEM (D6546; Sigma-Aldrich)
supplemented with 10% FBS (172021; Sigma-Aldrich), 2 mMl -glutamine
(25030-081; Gibco), and 50 µg/ml penicillin and streptomycin (15070-063;
Gibco) in a 5% CO2 incubator. For starvation treatment, cells were
washed twice with PBS and incubated in amino acid–free DMEM (048-33575;
Wako Pure Chemical Industries) without serum. Fip200 KO (Gan et al., 2006 (link)), Atg3KO (Sou et al., 2008 (link)), and
Atg5 KO (Kuma et al.,
2004 ) MEFs and ATG9A KO, ATG3 KO
(Tsuboyama et al., 2016 (link)), and TetON
GFP-STX17ΔNTD HeLa (Uematsu et al.,
2017 ) cells have been described previously. For bafilomycin
A1, Torin 1, or wortmannin treatment, cells were cultured with
100 nM bafilomycin A1 (B1793; Sigma-Aldrich), 250 nM Torin 1 (4247;
Tocris Bioscience), or 200 nM wortmannin (W1628; Sigma-Aldrich) for 2 h,
respectively. To visualize lysosomes, cells were cultured with 50 nM LysoTracker
red DND-99 (L-7528; Thermo Fisher Scientific) for 1 h.
supplemented with 10% FBS (172021; Sigma-Aldrich), 2 mM
(25030-081; Gibco), and 50 µg/ml penicillin and streptomycin (15070-063;
Gibco) in a 5% CO2 incubator. For starvation treatment, cells were
washed twice with PBS and incubated in amino acid–free DMEM (048-33575;
Wako Pure Chemical Industries) without serum. Fip200 KO (Gan et al., 2006 (link)), Atg3KO (Sou et al., 2008 (link)), and
Atg5 KO (
2004
(Tsuboyama et al., 2016 (link)), and TetON
GFP-STX17ΔNTD HeLa (
2017
A1, Torin 1, or wortmannin treatment, cells were cultured with
100 nM bafilomycin A1 (B1793; Sigma-Aldrich), 250 nM Torin 1 (4247;
Tocris Bioscience), or 200 nM wortmannin (W1628; Sigma-Aldrich) for 2 h,
respectively. To visualize lysosomes, cells were cultured with 50 nM LysoTracker
red DND-99 (L-7528; Thermo Fisher Scientific) for 1 h.
6
Immortalized Murine Striatal Cell Lines
7
Culturing Murine Monocytes (J774.2 Cell Line)
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Frozen murine monocytes (J774.2 Cell Line, Sigma–Aldrich, Dorset, UK) were cultured in DMEM (D6546, Sigma–Aldrich, Dorset, UK) containing 2 mM L‐glutamine (GE Healthcare Life Sciences, Buckinghamshire, UK) and 10% fetal bovine serum (FBS) (GE Healthcare Life Sciences, Buckinghamshire, UK). All cells were grown in an atmosphere of 5% CO2/95% air at 37 °C. Cells were dislodged into the media using a cell scraper prior to passaging. Cells were used at passage 5 in all experiments.
8
Murine Macrophage Cell Culture Protocol
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Cells were cultured using a murine macrophage cell line (RAW264.7, 91062702, Sigma-Aldrich, Gillingham, UK). Cells were maintained over permissive conditions in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; D6546, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Brazil, Thermo Fisher, Madrid, Spain), 2% L-glutamine (2 × 10−3 M, Sigma, St. Louis, MO, USA), and 1% penicillin G (100 U/mL, Sigma, St. Louis, MO, USA) at 37 °C in a humidified incubator with 5% CO2.
9
Isolation and Culture of Adrenal Medullary Cells
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Adrenal glands from WT and 3xTg mice were isolated and rapidly placed in ice-cold Locke solution ((in mM) 154 NaCl, 5.5 KCl, 3.6 NaHCO3, 10 Hepes, and 5.5 glucose (pH = 7.4)) after their sacrifice by cervical dislocation. The glands were fat trimmed, and the medullae of both glands were isolated from the capsule and the cortex and placed in a 1.5 mL tube containing 200 uL of Locke’s solution and papain (25 U/mL, Sigma-Aldrich) for tissue digestion at 37 °C for 26 min. This solution was exchanged with 1 mL of Dulbecco’s modified Eagle’s medium (DMEM; D6546, Sigma-Aldrich), repeating the exchange three times and finally leaving 120 μL of DMEM. Then, the medullae were mechanically digested by mincing them first with a 1 mL and then with a 200 μL micropipette tip. Finally, the residual medulla fragments were discarded and 10 μL drops of DMEM containing the cells were placed on poly-D-lysine-coated coverslips in 24-well plates. After a 1 h incubation (37 °C, water saturated, and 5% CO2 atmosphere), 500 μL of DMEM supplemented with 5% fetal bovine serum, 50 IU/mL penicillin, and 50 μg/mL streptomycin was added to each well. Experiments were conducted within the following 3 days.
10
Cell Culture and Starvation Protocol
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Cells were cultured in DMEM (D6546; Sigma-Aldrich) supplemented with 10% FBS (172012; Sigma-Aldrich), 50 U/ml penicillin, 50 µg/ml streptomycin (15070-063; Gibco), and 2 mM glutamine (25030-081; Gibco; regular medium) in a 5% CO2 incubator. For starvation treatment, cells were washed twice with PBS and cultured in Earle’s balanced salt solution (E2888; Sigma-Aldrich) or amino acid–free DMEM (048-33575; Wako) without FBS. For bafilomycin A1 treatment (B1793; Sigma-Aldrich), cells were cultured with 100 nM bafilomycin A1 for 2 h.
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