MEMbrane sheets were prepared as previously described (Wu et al., 2010 (link); Yong et al., 2018 (link)) with minor modifications. PTK2 cells were maintained in MEM (Invitrogen) supplemented with 10% (vol/vol) FBS at 37°C in 5% CO2. PTK2 cells were transfected using the Neon Electroporation kit (Invitrogen), and cells stably expressing TfR-pHluorin were maintained in the same medium supplemented with 0.5 mg/ml G418 (Invitrogen). Glass-bottom dishes (MatTek) or round coverslips (No. 1.5; Marienfeld-Superior) were washed with 1 M HCl and rinsed with PBS before being coated with poly-D-lysine and washed with sterile water overnight. The indicated cells were then plated and grown for 24–48 h until confluent. Basal MEMbrane sheets were generated by shearing the cells with several 1-s pulses of sonication in ice-cold cytosolic buffer.
Neon electroporation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Neon electroporation kit is a laboratory equipment designed for the delivery of genetic material into cells. It utilizes an electrical pulse to temporarily permeabilize the cell membrane, allowing the introduction of DNA, RNA, or other molecules into the cell interior.
Automatically generated - may contain errors
Lab products found in correlation
18 mm glass coated surfaces, by Marienfeld (3 mentions)
Poly l lysine (pll), by Merck Group (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
L 15 medium, by Thermo Fisher Scientific (3 mentions)
Anti cd3ε 16 0037, by Thermo Fisher Scientific (2 mentions)
Anti cd28 16 0289, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Scriptcap m7g capping system, by CellScript (2 mentions)
Scriptcap 2 o methyltransferase kit, by CellScript (2 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Human il 2, by Thermo Fisher Scientific (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
14 protocols using neon electroporation kit
1
Preparation of Basal Membrane Sheets
2
Jurkat T Cell Genetic Manipulation
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Jurkat T cells (ATCC Cat# TIB-152, RRID:CVCL_0367) and flotillin-1 and flotillin-2 knock-out Jurkat cell lines were cultured in RPMI 1640 medium (Gibco) supplemented with 10% (vol/vol) FBS, 2 mM l -glutamine and PenStrep (all from Invitrogen). Flotillin-1 and flotillin-2 knock-out Jurkat cell lines were generated using two guide RNAs each targeting the genomic DNA of flotillins 1 and 2 together with Cas9 expression plasmid. Transfected single cells were FACS sorted and seeded into 96-well plates and screened for flotillin1/2 knockout by western blot14 (link). Cells were transfected with 1 µg DNA per 200,000 cells, 18–20 h prior to imaging using the Neon electroporation kit (Invitrogen).
Before imaging, cells were incubated for 10 min at 37 °C on 18 mm glass-coated surfaces (Marienfeld) that were prepared by incubating with poly-l -lysine (Sigma) for 30 min at room temperature, then 1 µM anti-CD3ε (16-0037; eBioscience) and anti-CD28 (16-0289; eBioscience) antibodies for T cell activation. For live cell imaging, cells were imaged from 10 to 40 min after their deposition on the coverslips.
For immunostaining, cells were fixed with 3.7% EM-grade paraformaldehyde (C004, ProScitech) for 30 min at 37 °C. After fixation, cells were permeabilized with 0.15% triton-X100 (Sigma), blocked in 5% BSA and probed with primary and secondary antibodies sequentially.
Before imaging, cells were incubated for 10 min at 37 °C on 18 mm glass-coated surfaces (Marienfeld) that were prepared by incubating with poly-
For immunostaining, cells were fixed with 3.7% EM-grade paraformaldehyde (C004, ProScitech) for 30 min at 37 °C. After fixation, cells were permeabilized with 0.15% triton-X100 (Sigma), blocked in 5% BSA and probed with primary and secondary antibodies sequentially.
3
Activation and Genetic Modification of Human T Cells
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Primary human T cells were activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific) according to the manufacturer’s recommendations at a bead-to-cell ratio of 1:1 in the presence of 30 U/ml human IL-2 (PeproTech) for 72 h.
After bead removal, 106 cells were washed in DPBS and resuspended in 11 μl resuspension buffer R of the Neon electroporation kit (Invitrogen). 1 μl of the RNP mix was added to the cells immediately before electroporation. 10 μl of the mixture was electroporated with the Neon electroporation device at 1,600 V, 10 s with three pulses. The cells were transferred to 1 ml of prewarmed T-cell medium. Immediately afterwards, the virus solution was added at a multiplicity of infection (MOI) of 106 viral genomes per cell. For controls (TCRendo− T cells), the same volume of DPBS was added.
After bead removal, 106 cells were washed in DPBS and resuspended in 11 μl resuspension buffer R of the Neon electroporation kit (Invitrogen). 1 μl of the RNP mix was added to the cells immediately before electroporation. 10 μl of the mixture was electroporated with the Neon electroporation device at 1,600 V, 10 s with three pulses. The cells were transferred to 1 ml of prewarmed T-cell medium. Immediately afterwards, the virus solution was added at a multiplicity of infection (MOI) of 106 viral genomes per cell. For controls (TCRendo− T cells), the same volume of DPBS was added.
4
Transfection of Cell Lines
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COS-1 and HEK-293T were cultured in a 1:1 mixture of DMEM and Ham's F12 (DF12) media with 10% fetal bovine serum (FBS). HeLa and HAP1 cells were maintained in DMEM and IMDM, respectively, both with 10% FBS. Lipofectamine 2000 (Invitrogen) was used for the transfection of above cells. Human skin fibroblast cell lines from a mucolipidosis IV (TRPML1 KO) patient (clone GM02048) and a healthy control (clone GM05659) were obtained from the Coriell Institute for Medical Research (NJ, USA). Fibroblasts were transfected with a Neon electroporation kit (Invitrogen). Culture media were refreshed 18–24 h post-transfection, and cells were imaged 48 h post-transfection to allow sufficient recovery time following transfection.
5
Jurkat T Cell Flotillin Knockdown and Imaging
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Jurkat T cells (Clone E6.1) and flotillin-1 and flotillin-2 knock-out Jurkat cell lines were cultured in RPMI 1640 medium (Pan Biotech) supplemented with 10% (vol/vol) FCS (Gibco). Flotillin-1 and flotillin-2 knock-out Jurkat cell lines were generated as described in [15 (link)]. Cells were transfected with 1 μg DNA per 200,000 cells, 18–20 h prior to imaging using the Neon electroporation kit (Invitrogen).
Before imaging, cells were incubated for 10 min at 37 °C on 18-mm glass-coated surfaces (Marienfeld, #0,117,580) that were prepared by incubating with poly-L-lysine (Sigma, #P8920) for 30 min at room temperature, then 10 μg/ml anti-CD3ε (eBioscience, #16–0037) and anti-CD28 (eBioscience, #16–0289) antibodies for T cell activation. For live cell imaging, cells were imaged from 10 to 40 min after their deposition on the coverslips.
For imaging transferrin internalisation, transfected Jurkat T cells were activated for 5 min, transferred onto ice, media exchanged for fresh cold media with 25 μg/ml transferrin-Alexa488 (Jackson ImmunoResearch, #009–540-050) or transferrin-Alexa647 (Jackson ImmunoResearch, #009–600-050) added, incubated for 5 min, media exchanged twice with fresh cold media, then transferred to 37 °C and imaged.
Before imaging, cells were incubated for 10 min at 37 °C on 18-mm glass-coated surfaces (Marienfeld, #0,117,580) that were prepared by incubating with poly-L-lysine (Sigma, #P8920) for 30 min at room temperature, then 10 μg/ml anti-CD3ε (eBioscience, #16–0037) and anti-CD28 (eBioscience, #16–0289) antibodies for T cell activation. For live cell imaging, cells were imaged from 10 to 40 min after their deposition on the coverslips.
For imaging transferrin internalisation, transfected Jurkat T cells were activated for 5 min, transferred onto ice, media exchanged for fresh cold media with 25 μg/ml transferrin-Alexa488 (Jackson ImmunoResearch, #009–540-050) or transferrin-Alexa647 (Jackson ImmunoResearch, #009–600-050) added, incubated for 5 min, media exchanged twice with fresh cold media, then transferred to 37 °C and imaged.
6
Imaging Jurkat T-Cell Activation and Trafficking
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Jurkat T-cells (ATCC Cat# TIB-152, RRID:CVCL_0367) were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% (vol/vol) fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Invitrogen). T-cells were transfected with 1 µg DNA per 200,000 cells, 12–24 h prior to imaging using the Neon electroporation kit (Invitrogen).
Before imaging, cells were incubated for 10 min at 37°C on 18-mm glass-coated surfaces (Marienfeld) that were prepared by incubating with poly-l-lysine (Sigma) for 30 min at room temperature. Afterward coverslips were washed once with phosphate-buffered saline and incubated with 1 µM anti-CD3ε (16-0037; eBioscience) and anti-CD28 (16-0289; eBioscience) antibodies overnight at 4°C for T-cell activation. Cells were imaged from 10 to 40 min after their deposition on the coverslips.
For transferrin internalization, transfected Jurkat T-cells were activated as above and placed under the microscope. Alexa 647–conjugated transferrin (Jackson ImmunoResearch, USA) was added to T-cells 5 min after activation at 25 µg/ml (5 µl/ml), incubated for a further 10 min, and imaged.
Before imaging, cells were incubated for 10 min at 37°C on 18-mm glass-coated surfaces (Marienfeld) that were prepared by incubating with poly-l-lysine (Sigma) for 30 min at room temperature. Afterward coverslips were washed once with phosphate-buffered saline and incubated with 1 µM anti-CD3ε (16-0037; eBioscience) and anti-CD28 (16-0289; eBioscience) antibodies overnight at 4°C for T-cell activation. Cells were imaged from 10 to 40 min after their deposition on the coverslips.
For transferrin internalization, transfected Jurkat T-cells were activated as above and placed under the microscope. Alexa 647–conjugated transferrin (Jackson ImmunoResearch, USA) was added to T-cells 5 min after activation at 25 µg/ml (5 µl/ml), incubated for a further 10 min, and imaged.
7
Jurkat T Cell Cultivation and Transfection
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E6.1 Jurkat T cells (a gift from Brian C. Schaefer, Uniformed Services University, MD, USA) were grown in RPMI medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin at 37°C in a CO2 incubator. Transfections were performed with cells using 1 µg of plasmid by electroporation using a Neon electroporation kit (Thermo Fisher Scientific). Prior to imaging, cells were transferred to CO2 independent L-15 medium (Fisher Scientific). Cells tested negative for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza).
8
CRISPR-Cas9 Knockout of PRKAA1 in Jurkat Cells
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PRKAA1, human AMPKα1 (4077757 seq G*U*U*GGCAAACAUGAAUUGAC) sgRNA was purchased from Synthego. The CRISPR/Cas9 electroporation mix, containing 100 uM PRKAA1 sgRNA in TE buffer and 1.0 mg/mL Cas9 mRNA (TriLink L7606), was incubated for 10 min, at room temperature, to allow RNP complexes to form. Jurkat cells, washed in PBS and resuspended in R buffer (Neon electroporation kit) at 2 × 107 cells/mL, were added to the RNP solution and electroporated at 1325 V using 3 pulses at 10 ms intervals using a Neon Electroporator (ThermoFisher, Waltham, MA). The suspension was incubated for 48 h in an antibiotic-free culture medium before being transferred to a regular growth medium. Clonal populations, derived using limit dilution, were expanded and gene knockout was confirmed with TIDE [47 (link)] and Western blot analyses.
9
VEE Replicon Plasmid DNA Preparation
10
Generation of VEE-mCherry Replicon RNA
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Wildtype VEE replicon RNA was prepared as described in Wrobleska et al.17 (link),26 (link). mCherry was amplified by two round PCR with the primers YL-mCherry-ClaI-F, YL-mCherry-R1, and YL-mCherry-ClaI-F, YL-mCherry-SphI-R2 (Supplementary Table 2 ). Fragments from the second round PCR were cloned into the VEE replicon construct43 (link),44 (link) to obtain plasmids encoding the wildtype VEE-mCherry construct.
Replicon RNAs were in vitro transcribed (IVT) from the templates of linearized VEE-constructs above using the MEGAscript™ T7 Transcription Kit (ThermoFisher) following the manufacturer’s instructions. Resulting replicon RNAs were capped and methylated using the ScriptCap™ m7G Capping System and ScriptCap™ 2′-O-Methyltransferase Kit (Cellscript) according to the manufacturer’s instructions. RNA purity was assessed by gel electrophoresis.
In vitro transfections were carried out using 1 µg RNA for per 200,000 cells using the NEON electroporation kit (ThermoFisher) following the manufacturer’s instructions.
Replicon RNAs were in vitro transcribed (IVT) from the templates of linearized VEE-constructs above using the MEGAscript™ T7 Transcription Kit (ThermoFisher) following the manufacturer’s instructions. Resulting replicon RNAs were capped and methylated using the ScriptCap™ m7G Capping System and ScriptCap™ 2′-O-Methyltransferase Kit (Cellscript) according to the manufacturer’s instructions. RNA purity was assessed by gel electrophoresis.
In vitro transfections were carried out using 1 µg RNA for per 200,000 cells using the NEON electroporation kit (ThermoFisher) following the manufacturer’s instructions.
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