The human aortic vascular smooth muscle cell line (T/G HA-VSMC), which was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), was a gift by Dr. Chen Jian’s Lab. The human umbilical vein endothelial cell line (PUMC-HUVEC-T1) and human monocytic cell line (THP-1) were purchased from National Infrastructure of Cell Line Resource of China (Beijing, China). T/G HA-VSMC and PUMC-HUVEC-T1 cell lines were maintained in high-glucose DMEM (Life Technologies, Gibco) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Gibco), MEM Non-essential amino acids (MEM NEAA 100×, Life Technologies, Gibco), penicillin (100 U mL−1, Gibco, USA) and streptomycin (100 μg mL−1, Gibco, USA). THP-1 cells were maintained in RPMI 1640 Medium (Caisson Labs) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U mL−1) and streptomycin (100 μg mL−1). The cell lines were cultured in cell incubator (37 °C, 5% CO2) (Thermo Fisher Scientific). Human DiI-Acetylated LDL and LDL were purchased from Yeasen (Shanghai, China).
Mem neaa 100
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
MEM NEAA (100x) is a laboratory reagent that provides a concentrated solution of non-essential amino acids for cell culture applications. It is designed to be diluted and added to cell culture media to supplement the amino acid composition.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Fortecortin, by Merck Group (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Caisson (1 mentions)
Cell incubator, by Thermo Fisher Scientific (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Caisson (1 mentions)
Streptomycin, by Caisson (1 mentions)
T g ha vsmc, by American Type Culture Collection (1 mentions)
Chloroquine, by Merck Group (1 mentions)
35 mm culture dishes, by BD (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
12 protocols using mem neaa 100
1
Vascular cell culture protocol
2
HeLa Cell Culture and Transfection
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HeLa CCL-2 (ATCC) cells were grown in DMEM medium (Life Technologies) with 10% FBS (Life Technologies), 0.5 mM L-glutamine (Life Technologies), 1% MEM NEAA 100× (Life Technologies), and 1% penicillin/streptomycin (10,000 U/ml; Life Technologies) at 37°C and 5% CO2. HeLa cells were transfected using FuGENE 6 transfection reagent (Promega) according to the manufacturer’s instructions. For cryo-ET, ∼25,000 cells were seeded in 35-mm culture dishes (BD Falcon) containing four pre-treated EM grids 24 h before transfection. For Western blot experiments, cells were plated in 6 cm dishes 24 h before transfection (250,000 cells/well). To inhibit autophagy, cells were treated with 50 μM chloroquine in water (Sigma-Aldrich). HeLa cells were harvested 24–48 h after transfection.
3
Detailed Cell Culture Protocols
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HeLa cells were grown in Gibco™ DMEM Medium (1x Dulbecco’s Modified Eagle Medium; ThermoFisher Scientific, Cat. No. 31885-023) including 10% Fetal Bovine Serum (Biochrom AG; Cat. No. S0415), 1% MEM NEAA (100x) (Minimum Essential Medium Non-Essential Amino Acid; ThermoFisher Scientific; Cat. No. 11140-035) and 1% Penicillin-Streptomycin (Sigma; Cat. No. P0781) overnight at 37 °C. Jurkat cells are grown in Gibco™ RPMI Medium 1640 (1x) (Thermo Fisher Scientific; Cat. No. 31870-025) including 10% Fetal Bovine Serum (Biochrom AG; Cat. No. S0415), 1% MEM NEAA (100x) (Minimum Essential Medium Non-Essential Amino Acid; ThermoFisher Scientific; Cat. No. 11140-035) and 1% Penicillin-Streptomycin (Sigma; Cat. No. P0781). After aspiration of medium, and PBS wash, HeLa cells are incubated in Trypsin/EDTA (Sigma; Cat. No. T3924) for 5–10 min at 37 °C followed by addition of medium to stop trypsination of cells. After centrifugation (Hettich Rotina 380 R; 5 min × 300 g) of HeLa or Jurkat cells and an additional washing in PBS, cells are counted in Counting Slides (BIO RAD; Cat. No. 145-0011) in TC20 Automated Cell Counter (BIO RAD) by staining with trypan-blue (BIO RAD; Cat. No. 145-0013). In general, a fraction of >80% of viable cells were determined. After counting, cells were diluted to the intended cell number per sample.
4
Peptide Synthesis and Characterization
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OymaPure,
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Cys(Trt)-OH Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Gln(Trt)-OH,
and Fmoc-Phe-Wang resin were purchased from Novabiochem. N-Ethyldiisopropylamine (DIPEA), piperidine (≥99.5% for peptide
synthesis), and trifluoroacetic acid (TFA, ≥99.9%) were obtained
from Carl Roth. Dimethylformamide (DMF for peptide synthesis), diethyl
ether, and dimethyl sulfoxide (DMSO, ≥99.97+%) were purchased
from Acros Organics. Acetonitrile (HPLC grade) was purchased from
Fisher Scientific. Syringe filters Minisart SRP (0.20 μm) were
obtained from Sartorius. Glass coverslips (24 × 50 mm) were obtained
from Hirschmann and Glass coverslips (Ø = 13 mm) were purchased
from Fisher Scientific. ITO-coated glass slides for scanning electron
microscope (SEM) (15 × 20 mm) were obtained from Ossila and for
MALDI-MSI (25 × 75 mm) were purchased from Bruker Daltonics.
LE Agarose was obtained from Biozym Scientific. A549 cells, Dulbecco’s
Modified Eagle’s Medium (DMEM, 4.5 g/L glucose/glutamine),
penicillin/streptavidin, fetal bovine serum (FBS), and minimum essential
medium non-essential amino acids (MEM NEAA, 100×) were purchased
from Thermo Fisher Scientific. The ProteoStat Amyloid Plaque detection
Kit was purchased from Enzo Life Sciences, Inc. α-Cyano-4-hydroxycinnamic
acid (HCCA) was purchased from Sigma-Aldrich.
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Cys(Trt)-OH Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Gln(Trt)-OH,
and Fmoc-Phe-Wang resin were purchased from Novabiochem. N-Ethyldiisopropylamine (DIPEA), piperidine (≥99.5% for peptide
synthesis), and trifluoroacetic acid (TFA, ≥99.9%) were obtained
from Carl Roth. Dimethylformamide (DMF for peptide synthesis), diethyl
ether, and dimethyl sulfoxide (DMSO, ≥99.97+%) were purchased
from Acros Organics. Acetonitrile (HPLC grade) was purchased from
Fisher Scientific. Syringe filters Minisart SRP (0.20 μm) were
obtained from Sartorius. Glass coverslips (24 × 50 mm) were obtained
from Hirschmann and Glass coverslips (Ø = 13 mm) were purchased
from Fisher Scientific. ITO-coated glass slides for scanning electron
microscope (SEM) (15 × 20 mm) were obtained from Ossila and for
MALDI-MSI (25 × 75 mm) were purchased from Bruker Daltonics.
LE Agarose was obtained from Biozym Scientific. A549 cells, Dulbecco’s
Modified Eagle’s Medium (DMEM, 4.5 g/L glucose/glutamine),
penicillin/streptavidin, fetal bovine serum (FBS), and minimum essential
medium non-essential amino acids (MEM NEAA, 100×) were purchased
from Thermo Fisher Scientific. The ProteoStat Amyloid Plaque detection
Kit was purchased from Enzo Life Sciences, Inc. α-Cyano-4-hydroxycinnamic
acid (HCCA) was purchased from Sigma-Aldrich.
5
Detailed Antibody Staining Protocol
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Antibodies used in this study included: anti‐CD3‐fluorescein isothiocyanate (FITC) (clone UCHT1, cat. no. 21620033; ImmunoTools, Friesoythe, Germany), anti‐CD14‐allophycocyanin (clone MEM‐15, cat. no. 21279146, ImmunoTools), rabbit polyclonal immunoglobulin (Ig)G (C‐20) anti‐human NF‐κB primary antibody (cat. no. SC‐372R; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Alexa Fluor 594, goat anti‐rabbit IgG (H+L) secondary antibody (cat. no. R37117; Molecular Probes, Invitrogen, Carlsbad, CA, USA), biotin‐labeled phalloidin (cat. no. B7474; ThermoFisher, Fremont, CA, USA), streptavidin APC e‐fluor 780 (cat. no. 47‐4317‐82; ThermoFisher) and 4′,6‐diamidino‐2‐phenylindole (DAPI) nuclear stain (cat. no. 00‐4959‐52; ThermoFisher); RPMI‐1640 complete medium supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, 100 µg/ml streptomycin, 1 M HEPES buffer solution, 100 mM sodium‐pyruvate, minimum essential medium non‐essential amino acids (MEM NEAA) (×100) (all from gibco , Life Technologies, Carlsbad, CA, USA) and 2‐mercaptoethanol (Sigma, St Louis, MO, USA). Mycobacterial antigens used included: the 6‐kDa early secreted antigenic target (ESAT‐6) (15mer overlapping custom made peptides, Genscript, a kind gift from Dr Even Fossum) and ds‐Red fluorescent protein‐expressing bacille Calmette–Guérin (BCG) strain Myc3305 (provided by Dr Brigitte Gicquel) [27 ].
6
Culturing Common Cell Lines
7
Culturing Human Colorectal and Kidney Cells
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Human rectal tumor 18G cells (HRT-18G, American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in Dulbecco’s minimum essential medium containing Glutamax (DMEM + Glutamax; Gibco, Gaithersburg, MD, USA) and supplemented with 5% heat-inactivated Fetal Bovine Serum (FBS, PAN Biotech, Aidenbach, Germany), 100 μg/mL streptomycin, and 100 IU/mL penicillin (Gibco). The human embryonal kidney (HEK) cell line 293-LTV (LTV-100; Cellbiolabs, San Diego, CA, USA) was maintained in Dulbecco’s minimum essential medium containing Glutamax (DMEM + Glutamax; Gibco) and was additionally supplemented with 10% FBS (PAN Biotech), 1% minimum essential medium non-essential amino acids (MEM NEAA (100×), Gibco), 100 μg/mL streptomycin, and 100 IU/mL penicillin (Gibco). Both cell lines were cultured at 37 °C in a humidified incubator with 5% CO2.
8
Isolation of Primary Human Hepatocytes
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Tissue samples for PHH isolation were obtained from macroscopically tumor-free areas of resected livers (Table 1 ; D1–4 and DH2–10). PHHs were isolated as described previously [19 (link),20 (link),21 (link)] by a two-step EGTA/collagenase perfusion technique. Cells were pooled, washed with phosphate-buffered saline (PBS, Gibco, Paisley, UK) and resuspended in PHH culture medium (William’s Medium E, GlutaMAX™ (WME, Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS, Merck Biochrom, Berlin, Germany), 100 U/100 μM penicillin/streptomycin, 1% nonessential amino acids (MEM NEAA 100×), 15 mM HEPES, 1 mM sodium pyruvate (all provided by Gibco, Paisley, UK), 1 μg/mL dexamethasone (Fortecortin®, Merck, Darmstadt, Germany) and 80 IU/l human insulin (Lilly Deutschland GmbH, Bad Homburg, Germany/Sanofi-Aventis, Frankfurt am Main, Germany). Cell count and viability were determined using a Neubauer chamber with the trypan blue exclusion technique (Sigma-Aldrich, St. Louis, MO, USA).
9
Culture and Maintenance of Cell Lines
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The primary normal cells HMVEC-dBlAd-Adult dermal blood microvascular (CC-2811) and human renal proximal tubule cells—(CC2553) were purchased from Lonza (Switzerland) and grown in EGM 2MV bullet kit-(cc4147), and REGM bullet kit-(cc3191) media respectively, as recommended by the supplier. 3T3 mouse fibroblasts, human HL-60, NB4 and murine A20 leukemic cells (purchased from ATCC) were grown in RPMI 1640, 10% FBS, antibiotics and 2 mM l -glutamine (Biological Industries, Bet Haemek, Israel). Mouse MOPC 315.BM cells (kindly provided by Prof. Bjarne Bogen; University of Osla, Norway) were grown in RPMI 1640 GlutaMAX (Gibco) supplemented with 1% MEM NEAA 100× (Gibco), 1% sodium pyruvate (Gibco), 0.005% 1 M I-thioglycerol (Sigma), 0.03% Gensumycin 40 mg/ml (Sanofi Aventis) and 10% Fetal Bovine Serum (Gibco). Cells were cultured at 37 °C in 5% CO2 and passaged at 3- to 4-day intervals at sub-confluency. Murine cell suspensions of heart and kidney were prepared from Balb/c mice by mincing the tissue into PBS. The suspension was passed through a 70 µm mess and the cells washed thrice in PBS.
10
Hepatocyte and Kupffer Cell Culture
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The hepatocyte culture medium was based on Williams' Medium E with GlutaMAX (Gibco, Paisley, UK), supplemented with 10% FCS (Gibco), 32 mU/mL Insulin (Sanofi Aventis, Frankfurt am Main, Germany), 15 mM HEPES, 0.1 mM MEM NEAA (100×), 1 mM pyruvate (all by Gibco), and 1 mg/L dexamethasone (Fortecortin, Merck, Darmstadt, Germany).
KC culture medium was based on RPMI low glucose (GE Healthcare, Pasching, Austria) supplemented with 10% FCS, 1% L-glutamine, and 6.3 mM N-acetyl-L-cysteine (all by Gibco). KC starvation medium was based on RPMI low glucose supplemented with 1% L-glutamine. All media were supplemented with 100 U/100 μM penicillin/streptomycin (Gibco) prior to use.
PBS was purchased from Gibco. Percoll, Trypan Blue, and Hanks Balanced Salt Solution (HBSS) were provided by Biochrom (Berlin, Germany). All other chemicals were purchased from Sigma (Munich, Germany), if not stated differently.
KC culture medium was based on RPMI low glucose (GE Healthcare, Pasching, Austria) supplemented with 10% FCS, 1% L-glutamine, and 6.3 mM N-acetyl-L-cysteine (all by Gibco). KC starvation medium was based on RPMI low glucose supplemented with 1% L-glutamine. All media were supplemented with 100 U/100 μM penicillin/streptomycin (Gibco) prior to use.
PBS was purchased from Gibco. Percoll, Trypan Blue, and Hanks Balanced Salt Solution (HBSS) were provided by Biochrom (Berlin, Germany). All other chemicals were purchased from Sigma (Munich, Germany), if not stated differently.
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