24 hours before transfection, HEK293T cells were cultured in 10 cm cell culture dishes (Corning) or polylysine-coated 18 mm glass coverslips (VWR). One hour before transfection, media was changed to growth media supplemented with 0.6 mM 4-azido-l -phenylalanine. mGluR plasmids as described above and pIRE4-Azi plasmid (pIRE4-Azi was a gift from Irene Coin, Addgene plasmid # 105829) were co-transfected (1:1 w/w) into cells using Transporter 5 (Polysciences) or Lipofectamine 3000 (Invitrogen) (total plasmid: 1.5 μg/18 mm coverslip). For YADA/WT heterodimer experiments, SNAP-548UAA(WT), pIRE4-Azi and 548UAA-YADA were co-transfected (1:1:1 w/w/w) using Transporter 5 (Polysciences) (total plasmid: 2 μg/18 mm coverslip). The growth media containing 0.6 mM 4-azido- l -phenylalanine was refreshed after 24 hours and the cells were grown for another 24 hours (total 48 hours expression). On the day of the experiment, 10 minutes before labeling, supplemented growth media was removed and cells were washed twice by extracellular buffer solution containing (in mM): 128 NaCl, 2 KCl, 2.5 CaCl2, 1.2 MgCl2, 10 sucrose, 10 HEPES, pH=7.4 and were kept in growth medium without 4-azido-l -phenylalanine. Before the addition of labeling solution (below), cells were washed once with extracellular buffer solution.
Transporter 5
Manufactured by Polysciences
Sourced in United Kingdom
The Transporter 5 is a versatile laboratory equipment designed to transfer samples or materials between different locations or experimental setups. It features a stable platform, adjustable speed controls, and a secure clamping system to ensure safe and reliable transportation of samples.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
10 cm cell culture dishes, by Corning (2 mentions)
Polylysine coated 18 mm glass coverslips, by Avantor (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
X tremegene hp, by Roche (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 anti mouse thy 1.1 antibody, by BioLegend (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Tri reagent, by Zymo Research (1 mentions)
Fugene6, by Polysciences (1 mentions)
Bovine serum albumin bsa, by Gemini Bio (1 mentions)
Nupage sample buffer, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein g beads, by Thermo Fisher Scientific (1 mentions)
9 protocols using transporter 5
1
Genetically Encoded Fluorescent Labeling
2
Genetically Encoded Fluorescent Labeling
3
Overexpression and Knockdown of DNA Repair Proteins in Human Cell Lines
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HEK293T and HeLa cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (HyClone), 100 U/mL penicillin G (Life Technologies), and 100 mg/mL streptomycin (Life Technologies) in a humidified atmosphere of 5% CO2 at 37°C. Plasmids expressing HA-tagged PCNA constructs were cloned into the pcDNA5 FRT-TO, and S-tagged PCNA constructs were cloned into the pcDNA3 mammalian expression vector. The V5(GKPIPNPLLGLDST) epitope-tagged FEN1 and LIG1 constructs were cloned into a pcDNA5 FRT-TO mammalian expression vector. Transfection of plasmid DNA was performed using X-tremeGENE™ HP (#6366546001, Roche) or Transporter 5 (#26008–5, Polysciences) and siRNAs were transfected using RNAiMAX (#13778500, Thermo Fisher). Cells were analyzed 48 h after transfection.
4
Cell Culture Protocols for Receptor Studies
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HEK293T cells (passage 7–20, ATCC), embryonic fibroblasts (Fq11 cells) derived from Gq/11 knockout mice69 (link), and Gq/11/12/13-deleted HEK293 cells65 (link),66 (link) were cultured at 37 °C with humidified air containing 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, high glucose, Gibco) containing 10% fetal bovine serum (FBS, Gibco), penicillin, streptomycin, and sodium pyruvate. Fq11 cells were kindly provided by Dr. Patricia Hinkle (University of Rochester, Rochester, NY)69 (link), and Gq/11/12/13-deleted HEK293 cell70 (link) was a generously gift from Dr. Asuka Inoue (Tohoku University, Miyagi, Japan). Jurkat T cells (E6.1, ATCC) were maintained in RPMI 1640 (Corning) with 10% FBS, penicillin, streptomycin, and L-glutamine at a density of 0.5–1.5 cells ml−1. Transfections were carried out by using either Lipofectamine2000™ (Invitrogen) or Transporter™ 5 (Polysciences) following manufacturers’ manuals.
5
Enrichment of Thy1.1-Expressing Cells
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3×106 293T cells were seeded to 10 cm petri dishes and incubated overnight. Cells were transfected with 8 μg of plasmid DNAs (pCMV-Thy1.1-F2A-dsGFP/muSOX/SOX/vhs/BGLF5) using 48 μl Transporter 5 (Polysciences #26008) and 1ml Opti-MemI (Gibco #31985070). After 24 hours, cells were resuspended in 5 ml staining buffer [1x PBS Gibco # 10010023 supplemented with 2mM ethylenediaminetetraacetic acid (Sigma) and 0.5% FBS (Gibco)] and mouse Thy1.1-expressing cells were magnetically enriched with CD90.1 MicroBeads (Mlitenyi #130-121-273). Cells were stained with Alexa Fluor 647 anti-mouse Thy-1.1 Antibody (BioLegend; clone OX-7) and enrichment was confirmed with CytoFlex S (Beckman Coulter). 70~80% cells were positive for Thy1.1 after sorting and lysed with TRI reagent (Zymo Research #R2050–1) for RT-qPCR.
6
Visualizing Golgi Protein Trafficking
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For capturing release of the RUSH reporters from the Golgi, HeLa cells on coverslips were transfected with U21-SBP-GFP or SBP-GFP-CDMPR (FuGENE6 or Transporter5, Polysciences, Warrington, PA; cat. no. 26008). RUSH reporters U21-SBP-GFP or SBP-GFP-CDMPR were released from the ER with biotin (40 µM) for 20 min at 37°C and then fixed in 4% paraformaldehyde. Cells were then washed with phosphate-buffered saline (PBS) and permeabilized with 0.5% saponin + 3% bovine serum albumin (BSA; Gemini BioProducts, West Sacramento, CA; cat. no. 700-100P) before being labeled with primary and then Alexa Fluor–conjugated secondary antibodies. All steps were performed in 0.5% saponin + 3% BSA, with the exception of three final PBS washes. For colocalization of GGA3 with U21-SBP-GFP or SBP-GFP-CDMPR, cells were instead fixed in methanol for 15 min at −20°C. For colocalization of clathrin with internalized α-U21 and α-class I MHC, Zenon-647 IgG1 (Invitrogen Z25008) was used to label α-clathrin heavy chain (X22).
7
Pseudovirion Production for Hantavirus Research
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Pseudovirions were produced using the HTNV, SEOV, PUUV, DOBV, ANDV, SNV, and CHOCV DNA vaccine plasmids described above. HEK293T cells were seeded in T75 tissue culture flasks and transfected with the plasmid of interest using Transporter 5 (Polysciences Inc) at ∼80% confluency. After ∼18 h the transfection media was removed and the cells were infected with VSVΔG∗rLuc at a multiplicity of infection of ∼0.02 for 1 h at 37°C. The media was removed and fresh media was added, the flasks were then incubated at 32°C for 72 h. The supernatant from infected cells was collected and clarified by high speed centrifugation, followed by a PEG 8,000 precipitation with 3.2% salt. The PEG mixture is spun at10K xG for 45 min. The pellet was resuspended overnight in 1 mL TNE buffer, then filtered using a 0.45 μm filter, aliquoted and stored at 70°C.
8
Transfection Protocols for HEK293T Cells
9
Immunoprecipitation and Western Blot Analysis of Protein Complexes
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HEK293T cells were seeded to ~60% confluence in 100-mm dishes and incubated 1 day.
Each plasmid was then transfected with Transporter 5 (Polysciences) reagent according to the manufacturer's instructions. After 24 h incubation, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in buffer X (100 mM Tris-HCl pH8.0, 250 mM NaCl, 1 mM EDTA, 1% NP-40) with protease inhibitor cocktail (Merck, 11836170001), Benzonase (Enzynomics, M018S), and 5 mM MgCl 2 . We homogenized cell lysates by sonication and removed insoluble debris by centrifugation at 15000 rpm at 4ºC for 10 min.
Primary antibody (anti-Myc, 9E10, Santa Cruz) was added to the supernatant for overnight immunoprecipitation. The immunocomplexes were pulled down with dynabeads protein G beads (Invitrogen) and washed three times with buffer X. Samples were eluted with 2X
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
NuPAGE sample buffer (Invitrogen), then subjected to SDS-PAGE. For endogenous immunoprecipitation, we used ~80% confluent HEK293T cells and immunoprecipitated with each antibody (anti-MSH2, ab52266, abcam; anti-SMARCAD1, NB100-79835, Novus; anti-EXO1, ab95012, abcam).
Each plasmid was then transfected with Transporter 5 (Polysciences) reagent according to the manufacturer's instructions. After 24 h incubation, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in buffer X (100 mM Tris-HCl pH8.0, 250 mM NaCl, 1 mM EDTA, 1% NP-40) with protease inhibitor cocktail (Merck, 11836170001), Benzonase (Enzynomics, M018S), and 5 mM MgCl 2 . We homogenized cell lysates by sonication and removed insoluble debris by centrifugation at 15000 rpm at 4ºC for 10 min.
Primary antibody (anti-Myc, 9E10, Santa Cruz) was added to the supernatant for overnight immunoprecipitation. The immunocomplexes were pulled down with dynabeads protein G beads (Invitrogen) and washed three times with buffer X. Samples were eluted with 2X
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
NuPAGE sample buffer (Invitrogen), then subjected to SDS-PAGE. For endogenous immunoprecipitation, we used ~80% confluent HEK293T cells and immunoprecipitated with each antibody (anti-MSH2, ab52266, abcam; anti-SMARCAD1, NB100-79835, Novus; anti-EXO1, ab95012, abcam).
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