Human embryonic kidney 293 cells (HEK293) were seeded on 6-well plates at 2 × 105 cells/well in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1x GlutaMAX (Thermo Fisher) and 1% penicillin/streptomycin (Gibco) and incubated at 37 °C overnight in a humidified atmosphere with 5% CO2. The cells were transduced with AAV-anti-Scg3Fab, AAV-aflibercept or AAV-mCherry at 5 × 106 vg/mL. The medium was replaced with a serum-free 293SFM II medium (Thermo Fisher) the following day. Five days post-transduction, the conditioned medium was collected and concentrated using an Amicon® Ultra-4 Centrifugal Filter Unit (UFC801008, Millipore Sigma, St. Louis, MO, USA). An enzyme-linked immunosorbent assay (ELISA) was performed with pre-immobilized Scg3 (5 μg/mL, 100 μL/well, Sino Biological, Wayne, PA, USA), recombinant human VEGF (VEGF, 5 μg/mL, R&D Systems, Minneapolis, MN, USA) or bovine serum albumin (BSA, Sigma). Bound anti-Scg3 mAb and aflibercept were detected with biotin-conjugated anti-FLAG M2 mAb and horseradish peroxidase (HRP)-conjugated streptavidin (Sigma), followed by a colorimetric assay [27 (link)].
293 sfm 2 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
293 SFM II medium is a serum-free and protein-free cell culture medium designed for the growth and maintenance of 293 cells. It provides the necessary nutrients and growth factors to support the proliferation of 293 cells in suspension culture.
Automatically generated - may contain errors
Lab products found in correlation
Jetprime reagent, by Polyplus Transfection (2 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 4 centrifugal filter unit, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated streptavidin, by Merck Group (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Pei transfection reagent, by Polysciences (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Amicon centrifugal filter unit, by Merck Group (1 mentions)
0.2 μm syringe filter, by Pall Corporation (1 mentions)
7 protocols using 293 sfm 2 medium
1
Quantifying Anti-Scg3 and Aflibercept Production
2
Isolation and Concentration of Extracellular Vesicles
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The human embryonic kidney cell line HEK293sus [CRL-1573; American Type Culture Collection (ATCC)] was cultured in 293 SFM II medium (Thermo Fisher Scientific, MI, USA) supplemented with 2% GlutaMAX™-I (Thermo Fisher Scientific). Cells were grown until ≈70-80% confluence and then cultured in an EV-depleted medium for 48 h. EVs were collected from the resulting culture supernatant by ultracentrifugation to reduce heterogeneity caused by their size. The supernatant was centrifuged at 300 × g for 10 min, 2000 × g for 10 min, and 10000 × g for 30 min at 4 °C. After filtration with 0.22 μm membrane filter, and then ultracentrifuged at 120000 g for 100 min at 4 °C.
The EV pellets were washed with PBS by re-centrifugation under the same conditions. Protein concentrations were determined using BCA assay (Thermo Fisher Scientific). Protein concentrations of EVs were adjusted to 300 μg mL -1 .
The EV pellets were washed with PBS by re-centrifugation under the same conditions. Protein concentrations were determined using BCA assay (Thermo Fisher Scientific). Protein concentrations of EVs were adjusted to 300 μg mL -1 .
3
Production and Purification of MERS-CoV and SARS-CoV S1-Fc Proteins
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Plasmids encoding the MERS-CoV S1 domain (residues 1–747) and variants fused to the Fc region of the human IgG (MERS-CoV S1-Fc) were kindly provided by Dr. Bart Haagmans (Erasmus MC, Rotterdam, The Netherlands). Similarly, an S1-Fc expression plasmid was constructed for the SARS-CoV domain S1 subunit (residues 1–676) by using standard cloning techniques. The recombinant Fc-fusion proteins were expressed and purified as previously described [50 (link)]. Briefly, Fc-fusion proteins were expressed by transiently transfecting HEK-293T cells with the expression plasmids using polyethylenimine (PEI) transfection reagent (Polysciences, Warrington, PA, USA, 23966). Following an incubation time of 7 days in HEK-293T expression medium (293SFM II medium, Life Technologies, Carlsbad, CA, USA), the culture supernatants were harvested and clarified by centrifugation. The proteins were affinity purified using protein A sepharose beads (GE Healthcare, Chicago, IL, USA). Purified proteins were subjected to SDS–PAGE and visualized by Coomassie blue staining and Western blot analysis.
4
In Vitro Wound Healing Assay for Analyzing HRMVEC Migration
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HRMVEC migration was analyzed with in vitro wound healing assay, as described [22 (link),30 (link)]. Briefly, HRMVECs were cultured in 12-well plates until approximately 90–100% confluence. Cells were starved for 3 h in 293 SFM II medium (Life Technologies) supplemented with 2% fetal bovine serum (FBS). A sterile 200 µl tip was used to create a defined and clear scratch approximate 1 mm in width in each well. The dislodged cells were immediately removed by rinsing, and the remaining cells were supplemented with fresh 293 SFM II medium containing 2% FBS in the presence of HDGF, VEGF, or PBS. The migration of cells was monitored at 0 and 20 h as follows. At 0 h, at least six images per well were acquired with phase-contrast microscopy, and the average width of the original scratch (AW) was calculated. At 20 h, cells were incubated with 2 μM Calcein AM (Life Technologies) for 30 min at 37 °C and analyzed with fluorescence microscopy. At least six images were acquired for each well. The area percentage of the migrated cells was calculated using ImageJ software (NIH). Briefly, each image was cropped with the width of AW at 0 h. The area percentage of the migrated cells within the cropped image was calculated for each image with the threshold function of ImageJ software (NIH). The data of all images in the same well were averaged. We analyzed three wells for each group.
5
Quantifying ERK Activation in Endothelial Cells
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ERK activation was detected as described with modifications [6 (link)]. Briefly, HUVECs or HAECs were seeded in 6-well plates precoated with gelatin and cultured to ~90% confluence. Cells were preincubated in 293 SFM II medium (Life Technologies, Grand Island, NY) for 15 min x 3 times at 37°C to reduce the effect of other growth factors. Then the cells were incubated with HRP-3 for 10 min in 37°C and lysed in RIPA buffer (Pierce Biotechnology, Rockford, IL) containing phosphatase inhibitor cocktail (Roche). The lysates were analyzed by Western blot using antibody against phosphorylated ERK1/2 (pEKR1/2), ERK1/2 or β-actin (Cell Signaling, Danvers, MA), followed by horseradish peroxidase-labeled secondary antibody for chemiluminescence to detect the signals. Epidermal growth factor (EGF, Life Technologies) has been used in many previous studies of ERK1/2 activation and was included as a positive control.
6
Apoptosis-induced FLAG-protein secretion
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ABCF1-FLAG plasmid was transfected in HEK293T cells using jetPRIME reagents (Polyplus Transfection, Illkirch, France). After 48 h, the cells in 293SFM II medium (Life Technologies) were treated with or without etoposide for 16 h to induce apoptosis. The conditioned medium was collected from the apoptotic or healthy cells and centrifuged at 200 × g for 10 min. The cell-free supernatant was concentrated by filter concentrator units (Pierce Biotechnology, Rockford, IL; 9-kDa molecular weight cutoff) and analyzed by Western blotting using anti-FLAG M2 monoclonal antibody (Sigma-Aldrich).
7
Mesd-FLAG Release from Apoptotic HEK293 Cells
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