Hela and human skeletal muscle (hsSKM) cells were purchased from ATCC. HEK293A cells were purchased from Thermo Fisher. These cells were grown in DMEM (Biological industries, Israel) supplemented with 10% FBS (Biological industries, Israel) and 1% penicillin-streptomycin. Cells were maintained at 37°C and 5% CO2. Cells were transfected with polyethylenimine (PEI) or LipoGene 2000 Star Transfection Reagent (US Everbright). Mouse embryonic fibroblast (MEF, ATCC) cells were grown for at least six generations in Dulbecco’s modified Eagle’s medium (DMEM) for SILAC (Thermo Fisher) supplemented with L-lysine and L-arginine (light) or L-lysine-13C6 and L-arginine-13C6, 15N4 (heavy) (Thermo Fisher) as previously described (Wang J. and Nakamura F. 2019 (link)). 50 mg of each amino acids was added into every 500 mL DMEM for SILAC.
Lipogene2000 star transfection reagent
Manufactured by US Everbright
LipoGene2000 Star is a transfection reagent. It is designed to facilitate the delivery of nucleic acids, such as plasmids, siRNA, or mRNA, into mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (2 mentions)
Hek293a cells, by Thermo Fisher Scientific (1 mentions)
Dmem for silac, by Thermo Fisher Scientific (1 mentions)
L arginine and l lysine, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Sartorius (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
4 protocols using lipogene2000 star transfection reagent
1
Cell Culture and SILAC Labeling
2
CRISPR-mediated LARP4 knockout in HEK293A cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LARP4 KO HEK293A cells were generated by delivery of Cas9 and target-specific guide RNAs (gRNAs). Oligos encoding the gRNAs for LARP4 were designed using CRISPick (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design ) and the selected LARP4-specific gRNAs sequence, 5′-TAGACCGAGTACTGTTGGTG-3′ and 5′-TTGCGGCGGCGGGAACGATT-3′, were cloned into BbsI digested pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene plasmid ID: 42,230). Px330-LARP4 plasmids were transfected into HEK293A cells using LipoGene2000 Star transfection reagent (US Everbright) according to manufactures’ protocol. Briefly, HEK293A cells were seeded into a 24-well plate. After 24 h that the cells reached 60%–70% confluent, 1 μg px330-LARP4 plasmid was added to the well in the presence of LipoGene2000 Star transfection reagent. 72 h post-transfection, cells were then separated as single cells into a 96-well plate by serial dilution for another 7 days. Individual clones were expanded, and LARP4 protein expression was examined by immunoblotting.
3
Tracking Cell Migration in LARP4 Knockdown
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For random cell migration, HEK293A cells and LARP4-KD cells were seeded at 104 cells per well on 6-well plates. 24 h later, pAc-GFP-LARP4 WT and F277A plasmids were transfected into LARP4-KD cells using LipoGene2000 Star transfection reagent (US Everbright) according to manufactures’ protocol. After a further 24 h, images were acquired for 16 h at 1 frame/10 min at 37C using a ×10 Plan FL objective on an EVOS® FL Auto time lapse microscope with a monochrome and color camera. Cells were tracked using ImageJ (Plugin: Manual tracking) to obtain migration speed (μm/min). Cells that died, divided, or moved out of the frame were excluded from the analysis and tracking. The path of each cell was obtained as a track using ImageJ (Plugin: Chemotaxis tool).
4
Overexpression of ATG4a in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To overexpress ATG4a, ATG4a sequence was cloned into a lentiviral vector, pLenti-GIII- CMV-GFP-2A-Puro vector (cat. no. LV082668; Applied Biological Materials, Inc.). An empty vector was used as a negative control (NC). The sequencing primers of the ATG4a lentiviral vector were as follows: CMV sequencing primer, 5′-CGCAAATGGGCGGTAGGCGTG-3′; SV40 reverse sequencing primer, 5′-TAGTCAGCCATGGGGCGGAGA-3′.
Cells were transferred to 6-well plates at a cell density of 50–60% and cultured in MEM with 10% FBS for 24 h. Then, the medium was replaced with Opti-MEM (Thermo Fisher Scientific, Inc.) reduced-serum medium, to which 4.0 µg plasmid DNA and 10 µl LipoGene™ 2000 Star Transfection Reagent (US Everbright, Inc.) with 0.5 ml Opti-MEM were added; the mixture was incubated for 20 min at room temperature. The mixture in each plate was mixed with another 2 ml Opti-MEM and incubated under 5% CO2 in a 37°C incubator for 6 h; then, the medium was replaced with complete medium and cultured for another 48 h. Cells transfected with the ATG4a and empty vectors were termed the overexpression (OE)-ATG4a and NC groups, respectively. Total proteins were harvested 36 h after transfection, and other experiments were performed 24 h after transfection.
Cells were transferred to 6-well plates at a cell density of 50–60% and cultured in MEM with 10% FBS for 24 h. Then, the medium was replaced with Opti-MEM (Thermo Fisher Scientific, Inc.) reduced-serum medium, to which 4.0 µg plasmid DNA and 10 µl LipoGene™ 2000 Star Transfection Reagent (US Everbright, Inc.) with 0.5 ml Opti-MEM were added; the mixture was incubated for 20 min at room temperature. The mixture in each plate was mixed with another 2 ml Opti-MEM and incubated under 5% CO2 in a 37°C incubator for 6 h; then, the medium was replaced with complete medium and cultured for another 48 h. Cells transfected with the ATG4a and empty vectors were termed the overexpression (OE)-ATG4a and NC groups, respectively. Total proteins were harvested 36 h after transfection, and other experiments were performed 24 h after transfection.
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!