Lentivirus-based SARS-CoV-2 pseudovirus was generated by transfecting HEK293T cells with a luciferase-expressing lentiviral backbone plasmid, accessory plasmids (pHDM-Hgpm2, pHDM-tat1b, pRC/CMV-rev1b), and a plasmid encoding the SARS-CoV-2 spike protein with a 21-residue cytoplasmic tail deletion (Wuhan Hu-1 strain; GenBank: NC_045512 ). The neutralization activity of ACE2-GpA RBCs was measured using a modified version of a recently reported protocol.26 1.25 × 104 293T-ACE2 cells (provided by Dr. Jesse Bloom, Fred Hutchinson Cancer Research Center) were seeded per well on poly-l -lysine-coated 96-well plates (Corning Life Sciences) 18 h before infection. Serial dilutions of control and ACE2-GpA-RBCs were seeded in 400 μL of media (DMEM supplemented with 10% FBS and Pen-Strep) and incubated with 3 μL of lentiviral particles pseudotyped with the SARS-CoV-2 spike protein (5.5 × 107 relative light units [RLU]/mL) for 4 h on an orbital shaker (400 rpm) at 37°C in the presence of 10 μg/mL Polybrene. The lentiviral backbone of this SARS-CoV-2 pseudovirus system expresses luciferase to enable detection of infected cells. RBCs were spun down at 500 × g for 10 min and 100 μL of supernatant was transferred to the 96-well plate with the seeded 293T-ACE2 cells. Luminescence was measured after 48 h using a plate reader (Tecan).
Poly l lysine coated 96 well plate
Manufactured by Corning
Sourced in United States
Poly-l-lysine-coated 96-well plates are a type of laboratory equipment designed for use in various cell culture and biological applications. The plates are coated with the positively charged amino acid polymer poly-l-lysine, which enhances the attachment and growth of cells on the surface of the wells. This product provides a standardized substrate for consistent and reproducible cell-based experiments.
Automatically generated - may contain errors
Lab products found in correlation
In cell elisa kit, by Abcam (2 mentions)
Spectramax m2e, by Molecular Devices (2 mentions)
Cytation 5 imaging, by Agilent Technologies (1 mentions)
Optiplate, by PerkinElmer (1 mentions)
Alphalisa surefire ultra total syk assay kit, by PerkinElmer (1 mentions)
Alphalisa surefire ultra p syk tyr525 526 assay kit, by PerkinElmer (1 mentions)
Envision 2104 system, by PerkinElmer (1 mentions)
Af1828, by Abcam (1 mentions)
Quinpirole, by Merck Group (1 mentions)
Forskolin, by Merck Group (1 mentions)
Flexstation 3 reader, by Molecular Devices (1 mentions)
8 protocols using poly l lysine coated 96 well plate
1
SARS-CoV-2 Pseudovirus Neutralization Assay
2
Evaluating E-Liquid Cytotoxicity in HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells were plated on poly-L-lysine–coated 96-well plates (Corning, NY) at a density of 30,000 cells per well at t = 0 and incubated for 4 to 6 h to allow cells to adhere. AMs were plated as described above. Cells were exposed to 10 puffs of vaped e-liquids using a 4 s, 70 ml puff and incubated for 22 to 24 h. Media were then replaced with a modified Ringers solution containing calcein-AM (3 μM) and propidium iodide (3 μM) and incubated for 30 min to measure viability stain. Cells were then imaged using a Cytation5 imaging plate reader (BioTek). The normalized ratio of the average fluorescence intensity of calcein and propidium iodide was reported.
3
Vika Recombinase Cell Line Generation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Codon optimized Vika recombinase (GenBank: NZ_ACZN01000014.1 ZP_05884863) was synthesized with an N-terminal hemagglutinin (HA) epitope tag (YPYDVPDYA) and NLS (KKKRKV) (Thermo Fisher Scientific, Waltham, MA, USA). The synthesized fragment was cloned into the pFIV3.2CMVmcwtIRESneomycin lentiviral vector (The University of Iowa Viral Vector Core Facility, Iowa City, IA, USA), which uses the CMV promoter to drive expression of Vika. The HA epitope tag, NLS, and the Vika cDNA were synthesized directly adjacent without intervening linker sequences. Lentivirus was produced by The University of Iowa Viral Vector Core. 293 cells were transduced with Vika lentivirus, and transduced cells were selected by growth in medium containing 1 mg/mL G418. To establish clonal populations exhibiting high levels of Vika activity, Vika cells were transfected with a plasmid containing a CMV-vox-dsRed-vox-EGFP reporter cassette. Twenty-four hours post-transfection, cells were subjected to live cell FACS, gating on cells that were EGFP positive and dsRed negative (UIOWA flow Cytometry Core). Single cells were sorted into poly-L-lysine-coated 96-well plates (Corning Life Sciences, Tewksbury, MA, USA). Clones were expanded and assessed for excision activity and expression by reporter plasmid transfection and Western blot, respectively.
4
Astrocyte Response to Exosomal miR-146a
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BM-MSCs of passage three were used for culturing. BM-MSCs were seeded at a density of 1.5 × 104 cells/cm2 in a 15-cm dish. BM-MSCs were cultured in alpha-minimal essential medium with 15% FBS and 1% PS. When BM-MSCs reached 90% confluence, the dish was transfected with 10 nM miR-146a using Hiperfect transfection reagent. At 4 h after transfection, the dish was washed with PBS, and the conventional medium was replaced with 10 ml FBS-free alpha-minimal essential medium. After 24 h, the medium was collected, and exosomes were isolated with a MagCapture Exosome Isolation Kit PS. On day 1 after seeding astrocytes in poly-l -lysine-coated 96-well plates (Corning) at a density of 3.1 × 104/cm2, exosomes suspended in FBS-free DMEM/F-12 medium were added to astrocytes (0.4 µg/ml). After 24 h of adding exosomes, both miRNA and mRNA of astrocytes were extracted as described above. cDNA synthesis and real-time PCR were also performed as described above.
5
Quantification of TREM2-Mediated SYK Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In total, 2 × 10 4 iPSdMiG were plated per well in poly-L-lysine-coated 96 well plates (Corning) as described above. Then, 24 hr after plating, the cells were stimulated for 5 min with anti-TREM2 (5 μg/ml, R&D Systems #AF1828), corresponding IgG isotype control (5 μg/ml Abcam #ab224187) or left untreated. pSYK levels were detected by the AlphaLISA SureFire Ultra p-SYK (Tyr525/526) Assay Kit (PerkinElmer) and normalized to the values of tSYK using the AlphaLISA SureFire Ultra Total SYK Assay Kit (PerkinElmer) according to the two-plate assay protocol for adherent cells. All samples were measured as technical duplicates in 384-well OptiPlates (PerkinElmer). The plate was measured using the standard AlphaLISA settings on a PerkinElmer EnVision 2104 system. For the analysis, the technical duplicates were averaged and the pSYK signal was normalized to the tSYK signal. The Data was displayed as pSYK/tSYK relative to the WT signal.
6
Mitochondrial Biogenesis Assay in L6 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An equal number of L6 cells suspended in the growth medium was seeded on each well of a poly-L-lysine coated 96-well plate (Corning Inc. Corning, NY) and incubated overnight at 37°C in a CO2 incubator. The cells were treated with different concentrations of LI80020F4 for 72 h, with a repeat treatment every 24 h in a fresh growth medium. The VC culture wells received 0.2% DMSO (v/v). Mitochondrial biogenesis was determined using an in-cell ELISA kit (Abcam, Cambridge, UK), following the assay protocol provided by the manufacturer. Briefly, the treated cells were fixed with 4% paraformaldehyde and probed with primary antibodies against COX-1 (cytochrome c oxidase subunit I) and SDHA. The PBS-washed cells were reacted with alkaline phosphatase (AP) and horseradish peroxidase (HRP)-labeled secondary antibodies (Jackson Immuno Research, West Grove, PA) for the detection of SDHA and COX-1 expressions at 405 and 600 nm, respectively, in a multi-mode plate reader (Spectramax M2e, Molecular Devices, Sunnyvale, CA). The ratio between COX-I and SDHA expressions was calculated, and the percent (%) increase of mitochondrial biogenesis was determined in the treated cells over the VC cells.
7
Quantifying cAMP Modulation by D2 Receptors
8
Mitochondrial Biogenesis Assay in L6 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An equal number of L6 cells suspended in the growth medium was seeded on each well of a poly-L-lysine coated 96-well plate (Corning Inc. Corning, NY) and incubated overnight at 37°C in a CO2 incubator. The cells were treated with different concentrations of LI80020F4 for 72 h, with a repeat treatment every 24 h in a fresh growth medium. The VC culture wells received 0.2% DMSO (v/v). Mitochondrial biogenesis was determined using an in-cell ELISA kit (Abcam, Cambridge, UK), following the assay protocol provided by the manufacturer. Briefly, the treated cells were fixed with 4% paraformaldehyde and probed with primary antibodies against COX-1 (cytochrome c oxidase subunit I) and SDHA. The PBS-washed cells were reacted with alkaline phosphatase (AP) and horseradish peroxidase (HRP)-labeled secondary antibodies (Jackson Immuno Research, West Grove, PA) for the detection of SDHA and COX-1 expressions at 405 and 600 nm, respectively, in a multi-mode plate reader (Spectramax M2e, Molecular Devices, Sunnyvale, CA). The ratio between COX-I and SDHA expressions was calculated, and the percent (%) increase of mitochondrial biogenesis was determined in the treated cells over the VC cells.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!