The cells were cultured in mTeSR™1 (StemCell Technologies, Van-couver, BC, Canada) and coated with human vitronectin (Gibco, Gaithersburg, MD, USA). Cells were passaged by 1:10 with 0.02% EDTA (Sigma, Darmstadt, Germany). The 0.2 g EDTA was dissolved in 1 Liter Dulbecco’s phosphate-buffered saline (DPBS, Biological industries, Cromwell, CT, USA).
Ethylene diamine tetraacetic acid (edta)
Manufactured by Sartorius
Sourced in Israel, United States
EDTA (Ethylenediaminetetraacetic acid) is a chelating agent commonly used in laboratory applications. It is a multidentate ligand that can form stable complexes with various metal ions, including calcium, magnesium, and heavy metals. EDTA is utilized for activities such as metal ion analysis, sample preparation, and buffer formulations.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Sartorius (5 mentions)
100 mm culture dishes, by BD (2 mentions)
Human prpe cells, by Lonza (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Human vitronectin, by Thermo Fisher Scientific (1 mentions)
Mtesr1, by STEMCELL (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Sartorius (1 mentions)
Rtegm bulletkit, by Lonza (1 mentions)
100 mm dish, by BD (1 mentions)
Di potassium hydrogen phosphate trihydrate, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Passive lysis buffer 1, by Promega (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Tricine, by Merck Group (1 mentions)
Mgso4, by Merck Group (1 mentions)
Transit lt1, by Mirus Bio (1 mentions)
Rtegm bulletkit medium, by Lonza (1 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
10 protocols using ethylene diamine tetraacetic acid (edta)
1
Culturing Cells in mTeSR™1 with Vitronectin
2
Isolation and Characterization of Human pRPE and ASCs
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5.5 × 105 cells of human pRPE cells (Lonza) were plated in 100 mm culture dishes (Falcon) in retinal epithelial cell growth medium (RtEGM) Bulletkit (Lonza) and incubated at 37°C in a humidified 5% CO2 atmosphere. The medium was replaced twice weekly, and cells were passaged with 0.25% trypsin/0.05% EDTA (Biological Industries, Israel) upon reaching 90%confluence. Experiments were performed at passage three. OASCs and ABASCs (1.0 × 106 cells) at passage 3 were plated on a 100 mm dish (Falcon) and cultured in ASCs BulletKit™ Medium (Lonza). At 100% confluence, ASCs were washed with PBS and cultured with ASC serum free medium (Lonza) for 48 h before isolation of their respective medium, containing many released growth factors and cytokines. Both OASC-conditioned medium (OASC-CM) and ABASC-conditioned medium (ABASC-CM) were maintained at -80°C for further analysis using protein Array (RayBiotec).
3
Notch Signaling Pathway Activation Assay
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The activity of the different
ligands was tested using a luciferase reporter gene assay. Receiver
cells stably expressing either Notch1 or synNotch variants were co-transfected
in a 24-well plate using TransIT-LT1 (Mirus) or Lipofectamine 3000
(Thermo Fisher Scientific, for U2OS cells) with a Gal4-firefly luciferase
reporter (Andrawes et al.32 (link)) (300 ng) and
pRL-SV40 Renilla luciferase (10 ng). 24 h after transfection, the
cells were trypsinized and co-culture with cells stably expressing
the ligands. 48 h after plating, firefly luciferase and Renilla luciferase
activities were measured by luminometer (Veritas). Cells were lysed
with 100 μL/well passive lysis buffer 1× (Promega) for
10 min. 20 μL of each sample was used for luciferase activity
using filtered luciferase buffer including: 26 mg of (MgCO3)4 Mg(OH)2
(Sigma), 20 mM Tricine (Sigma), 0.1 mM EDTA (Biological Industries),
2.67 mM pH = 7.8 MgSO4 (Merck). For the luciferase reaction, we used
luciferase buffer supplemented with 0.4 mM ATP (Sigma), 26.6 mM DTT
(Sigma), Coenzyme A X0.8 (Sigma), and 0.4 mMd -Luciferin,
and for Renilla activity using filtered Renilla buffer including 80
mM di-potassium hydrogen phosphate trihydrate (Merck) and 20 mM potassium
dihydrogen phosphate for analysis (Merck). Notch activity is expressed
as a ratio of normalized luciferase by Renilla.
ligands was tested using a luciferase reporter gene assay. Receiver
cells stably expressing either Notch1 or synNotch variants were co-transfected
in a 24-well plate using TransIT-LT1 (Mirus) or Lipofectamine 3000
(Thermo Fisher Scientific, for U2OS cells) with a Gal4-firefly luciferase
reporter (Andrawes et al.32 (link)) (300 ng) and
pRL-SV40 Renilla luciferase (10 ng). 24 h after transfection, the
cells were trypsinized and co-culture with cells stably expressing
the ligands. 48 h after plating, firefly luciferase and Renilla luciferase
activities were measured by luminometer (Veritas). Cells were lysed
with 100 μL/well passive lysis buffer 1× (Promega) for
10 min. 20 μL of each sample was used for luciferase activity
using filtered luciferase buffer including: 26 mg of (MgCO3)4 Mg(OH)2
(Sigma), 20 mM Tricine (Sigma), 0.1 mM EDTA (Biological Industries),
2.67 mM pH = 7.8 MgSO4 (Merck). For the luciferase reaction, we used
luciferase buffer supplemented with 0.4 mM ATP (Sigma), 26.6 mM DTT
(Sigma), Coenzyme A X0.8 (Sigma), and 0.4 mM
and for Renilla activity using filtered Renilla buffer including 80
mM di-potassium hydrogen phosphate trihydrate (Merck) and 20 mM potassium
dihydrogen phosphate for analysis (Merck). Notch activity is expressed
as a ratio of normalized luciferase by Renilla.
4
Propagation of Human pRPE Cells
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5.5 × 105 cells of human pRPE cells (Lonza) were plated in 100 mm culture dishes (Falcon) in RtEGM BulletKit Medium (Lonza) and incubated at 37°C in a humidified 5% CO2 atmosphere. The medium was replaced twice weekly, and cells were passaged with 0.25% trypsin/0.1% EDTA (Biological Industries, Israel) upon reaching 90% confluence. Experiments were performed at passages 3–4.
5
Single-cell isolation for scRNA-seq
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Prior to dissociation, to focus on tissues that contribute to the embryo proper, the ectoplacental cone, parietal endoderm, and much of the extraembryonic ectoderm, were removed using fine forceps. For isolation of single cells for scRNA-seq, embryos were dissociated with 0.25% Trypsin-A, 0.02% EDTA (Biological Industries) solution for 5′ at 37°C, and resuspended in DMEM w/o phenol red (GIBCO) supplemented with 10% FBS (Biological Industries). Samples were run on a FACSAria-III flow cytometer (BD Biosciences) using the ‘index sort’ option to retain the spectral properties of each individual sorted cell.
6
Flow Cytometric Analysis of CAR-T Cells
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CAR-T samples were collected from cocultures following addition of Phosphate-buffered saline (PBS)+ 10 mM EDTA (Biological industries) for 1 min. Cells were stained and analyzed according to standard flow cytometry protocols at 4°C for 30 min in PBS (Sartorius)+ 2% FBS (Sigma-Aldrich) using a Celesta (BD) or Attune NxT (Invitrogen) cytometer. The Fluorescence-activated cell sorting (FACS) antibodies are listed in online supplemental table S1 . Dead cells were identified by 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) staining. Analysis was performed using FlowJo v10.8 Software (BD Life Sciences). For cell surface receptor quantification, cells were stained with Flag-APC, cMyc-FITC, Tet-PE or anti-trastuzumab+anti-human-APC. Quantum MESF beads and Quantum Simply Cellular beads (Bangs Laboratories) were used according to manufacturer recommendations. For iCAR quantification, tetramer Median Fluorescence Intensity (MFI) was calibrated using Jurkat cells stably expressing cMyc-tagged iCAR, that were stained with both Tetramer and cMyc. Cell-surface HLA-A2 and HER2 expression was quantified using anti-HER2 PE (Miltenyi Biotec, 1:5) and anti-HLA-A2 APC (Thermo Fisher, 1:10).
7
Cytotoxicity Evaluation of MeDZQ
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MeDZQ was a generous gift by Dr. Jonas Sarlauskas (Department of Xenobiotics Biochemistry, Institute of Biochemistry, Life Sciences Center, Vilnius University). It was synthesized according to a previously published procedure (Cameron and Gilles, 1968[7 ]; Winski et al., 1998[31 (link)]). Fetal bovine serum (FBS) was obtained from Life Technologies (USA), penicillin-streptomycin, trypsin and EDTA were purchased from Biological Industries (Israel). Dulbecco's modified Eagle medium (DMEM), acridine orange/ethidium bromide (AO/EB) mixture, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dicumarol (DIC), N,N'-diphenyl-p-phenylenediamine (DPPD), desferrioxamine (DESF), MAPK inhibitors PD098059, SP600125 and SB203580 were obtained from Sigma-Aldrich (USA). Stock solutions (20 mM) of all the inhibitors were prepared in dimethyl sulfoxide (DMSO). Anti-phospho-p38, anti-ERK and anti-JNK antibodies were purchased from Cell Signaling Technology (USA). Monoclonal antibodies for p38, phospho-ERK, and phospho-JNK detection were obtained from BD Biosciences (USA). Secondary goat anti-mouse and goat anti-rabbit antibodies (both conjugated with horseradish peroxidase) were purchased from Abcam (UK). Low melting-point agarose (LMPA) was purchased from Carl Roth (Germany), and standard agarose - from Thermo Fisher Scientific (USA).
8
Quantifying RPE Cell Death by Flow Cytometry
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Following rescue studies as described above, RPE cells at passage three were harvested with 0.25% trypsin/0.05% EDTA (Biological Industries). Cells (3 × 105) were collected by centrifugation at 500 g for 5 min, washed twice with PBS, and resuspended in 400 μl of PBS to which 1 μl of propidium iodide (1 mg/mL, Sigma) was added immediately before flow cytometry measurements. At least 10,000 events were collected and labeled, and fluorescent cells were detected by BD FACS CantoTM II cytometer (BD Pharmingen, USA). Analysis of cell death distribution was conducted by FCS Express 4 software (De Novo Software, Canada).
9
Assessing Targeted Delivery of fLuc mRNA in Colitis
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Colitis was induced in 12-weeks-old female C57BL/6 mice (Harlan laboratories, Israel) using dextran sodium sulfate (DSS). Mice were fed for 10 days with 1.5% (wt/vol) DSS in the drinking water. To assess the selectivity of fLuc mmRNA expression, fLuc mmRNA loaded LNPs were self-assembled with ASSET and αLy6C or isotype control antibodies and were injected intravenously on day 5 from DSS treatment (1 mg/kg). On day 6, Leukocytes were isolated from the spleen, isolated from red blood cells using an osmotic shock, passed through a 70 μm cell strainer and washed twice with PBS. Cells in PBS containing 2% fetal bovine serum and 2 mM EDTA (Biological Industries, Israel) were stained with 1:100 fluorescently labeled αLy6C antibodies (BioLegend, USA) for 30 min at 4 °C and sorted for Ly6C+ and Ly6C− cells by flow cytometry (FACSAria, BD, USA). Sorted cells were lysed and fLuc expression was assessed in each leukocyte population using Luciferase Assay System (Promega, USA) and Veritas Microplate Luminometer (Turner BioSystems).
10
Isolation and Culture of Human Adipose Stem Cells
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Human adipose tissue was harvested from 5 healthy patients with a mean age of 38 ± 4.3 and body mass index of 28.2 ± 3.9 who had abdominoplasty for aesthetic reasons at Tel Aviv Sourasky Medical Center. No metabolic diseases, HIV, hepatitis, or other systemic complications were reported from these patients.
The isolation and culture of ASCs were performed as previously described [15 (link)]. Briefly, 60 to 120 ml of the raw lipoaspirates was washed with phosphate-buffered saline (PBS) and enzymatically digested with 0.75% collagenase type I (Cat. no. C1639, Sigma) at 37°C for 1 hour. The digested lipoaspirates were centrifuged at 400 g for 15 minutes, and the pellet was resuspended and passed through a 100 μm mesh filter (Cat. no. 542000, EASYstrainer, Greiner Bio-One) to remove debris. Subsequently, 1 × 106 cells were plated in 100 mm culture dishes in ADSC medium and incubated at 37°C in a humidified 8% CO2 atmosphere [16 (link)]. The medium was changed twice weekly, and cells were passaged with 0.25% trypsin/0.1% EDTA (Biological Industries, Israel) upon reaching 90% confluency. Experiments were performed at passages 3–4.
The isolation and culture of ASCs were performed as previously described [15 (link)]. Briefly, 60 to 120 ml of the raw lipoaspirates was washed with phosphate-buffered saline (PBS) and enzymatically digested with 0.75% collagenase type I (Cat. no. C1639, Sigma) at 37°C for 1 hour. The digested lipoaspirates were centrifuged at 400 g for 15 minutes, and the pellet was resuspended and passed through a 100 μm mesh filter (Cat. no. 542000, EASYstrainer, Greiner Bio-One) to remove debris. Subsequently, 1 × 106 cells were plated in 100 mm culture dishes in ADSC medium and incubated at 37°C in a humidified 8% CO2 atmosphere [16 (link)]. The medium was changed twice weekly, and cells were passaged with 0.25% trypsin/0.1% EDTA (Biological Industries, Israel) upon reaching 90% confluency. Experiments were performed at passages 3–4.
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