LLC-MK2 cells were seeded in 15 μ-slide 8-well chambers (Ibidi, Germany) and transfected 1 day prior to imaging. Cells were transfected with intracellular markers for the plasma membrane (LCK-GFP; Addgene plasmid #61099), endoplasmic reticulum (pLV-ER GFP; Addgene plasmid #80069), Golgi apparatus (pLV-Golgi GFP; Addgene plasmid #79809), and mitochondria (HyPer-dMito; Evrogen). Control wells received TagRFP (Evrogen), while experimental wells received either full-length RFP-NS1-2, RFP-NS1-2(Δ157), or RFP-NS1-2(Δ176). Cells were imaged 24 h posttransfection on the DeltaVision LIVE high-resolution deconvolution microscope (GE Healthcare) using the 60×/1.4 Plan-Apo NA oil objective (Olympus), and acquired using a pco.edge sCMOS_5.5 camera. Images were acquired and deconvolved in SoftWoRx software. After the images were deconvolved, they were further processed in FIJI (ImageJ) to adjust for brightness/contrast and pseudocoloring (82 (link)).
Lck gfp
Manufactured by Addgene
Lck-GFP is a plasmid construct containing the gene encoding the lymphocyte-specific protein tyrosine kinase (Lck) fused to the green fluorescent protein (GFP). This fusion protein allows for the visualization and tracking of Lck localization and dynamics within living cells.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
15 μ slide 8 well chambers, by Ibidi (1 mentions)
Tagrfp, by Evrogen (1 mentions)
Plv er gfp, by Addgene (1 mentions)
Megfp lifeact 7, by Addgene (1 mentions)
Pegfp n1 human cofilin wt, by Addgene (1 mentions)
Pmrfp n1 human cofilin wt, by Addgene (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Pcmv5 src, by Addgene (1 mentions)
Mycoalert, by Lonza (1 mentions)
5 protocols using lck gfp
1
Visualizing intracellular organelles and RFP-NS1-2
2
Plasmid Verification and Utilization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All plasmids were acquired from Addgene and verified by sequencing using the associated primers on the Addgene website. The following plasmids were used: LCK-GFP (Addgene 61099), LCK-mScarlet-1 (Addgene 98821), pEGFP-N1 human cofilin WT (Addgene 50859), tdTomato-Lifeact-7 (Addgene 54528), mEGFP-Lifeact-7 (Addgene 54610), and pmRFP-N1 human cofilin WT (Addgene 50856).
3
Hippocampal Neuron Transfection with Lipofectamine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lipofectamine 2000 was used to transfect 3-week-old dissociated hippocampal cultures, using the following plasmids: mApple-F-tractin (Dr. H. N. Higgs), Lifeact-mRFP (Dr. Ray Truant), Lck-GFP (Addgene plasmid 61099), mem-mApple Empty vector and mem-mApple-INF2-shRNA (see above section about plasmid construction for INF2 RNA interference), GFP-INF2-wt (CAAX & non-CAAX) (Dr. H. N. Higgs), peGFP-N1 (Clontech), GFP-empty v., GFP-INF2-shRNA, GFP-INF2-CA (Dr. H. N. Higgs).
Fresh Neurobasal Medium was used to prepare the mixture of Lipofectamine and cDNA. In all, 1 µl of Lipofectamine and 1.5 µg of cDNA per 50 µl of NBM were mixed, incubated for 30 min and then added dropwise to 500 µl NBM in which neurons had been growing for 3 weeks. Washing was not required and neurons showed no overt signs of toxicity.
Fresh Neurobasal Medium was used to prepare the mixture of Lipofectamine and cDNA. In all, 1 µl of Lipofectamine and 1.5 µg of cDNA per 50 µl of NBM were mixed, incubated for 30 min and then added dropwise to 500 µl NBM in which neurons had been growing for 3 weeks. Washing was not required and neurons showed no overt signs of toxicity.
4
Engineered Src Constructs for Organelle Targeting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The constructs encoding hemagglutinin (HA)- or V5-tagged mouse Src (Src-HA and Src-V5) and HA- or V5-tagged mouse Src targeted to the mitochondria (mtSrc-HA and mtSrc-V5) were generated by amplification of the sequence of Src from pCMV5-Src (#13663, Addgene, MA, USA) and fused to HA or V5 with or without the mitochondrial leading sequence (MLS) of cytochrome c oxidase VIIIa, as described [16 (link)]. Constructs encoding FLAG-tagged human wild-type Src, constitutively active (CA) Src and kinase-dead (KD) Src fused to the MLS of the very long chain acyl-CoA dehydrogenase (named hereafter mtSrc-FLAG, mtSrc(CA)-FLAG and mtSrc(KD)-Flag, respectively) were obtained from Addgene (#44652, #44654 and # 44653, respectively). Src-V5 was targeted to the plasma membrane (pmSrc-V5) using the first 26 amino acids of Lck (derived from Lck-GFP obtained from Addgene #61099) as previously shown [42 (link), 43 (link)].
5
Cultivation and Characterization of Human Osteosarcoma Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human osteosarcoma cell line U2OS was grown in DMEM (high glucose, GlutaMAX) containing 10% FBS and penicillin–streptomycin (Thermo Fisher Scientific).
The U2OS FUCCI cells were kindly provided by Atsushi Miyawaki14 (link). These cells are endogenously tagged with two fluorescent proteins fused to the cell cycle regulators CDT1 (mKO2-hCdt1+) and geminin (mAG-hGem+). CDT1 accumulates during the G1 phase, whereas geminin accumulates in the S and G2 phases, allowing cell cycle monitoring. The cells were cultivated at 37 °C in a 5.0% CO2 humidified environment in McCoy’s 5A (modified) medium GlutaMAX supplement (Thermo Fisher Scientific, 36600021) supplemented with 10% FBS (VWR) without antibiotics.
U2OS cells stably expressing a membrane-targeted form of eGFP were generated by transfection with plasmid Lck-GFP (Addgene, 61099 (ref. 37 (link))) and culturing in selection medium (DMEM medium containing 10% FBS, penicillin–streptomycin and 400 μg ml−1 of Geneticin) under conditions of limited dilution to yield single colonies. A clonal cell line with homogenous and moderate expression levels of Lck-eGFP at the plasma membrane was established from a single colony.
All cell lines were tested for mycoplasma (MycoAlert, Lonza) and authenticated by STR profiling (IdentiCell).
The U2OS FUCCI cells were kindly provided by Atsushi Miyawaki14 (link). These cells are endogenously tagged with two fluorescent proteins fused to the cell cycle regulators CDT1 (mKO2-hCdt1+) and geminin (mAG-hGem+). CDT1 accumulates during the G1 phase, whereas geminin accumulates in the S and G2 phases, allowing cell cycle monitoring. The cells were cultivated at 37 °C in a 5.0% CO2 humidified environment in McCoy’s 5A (modified) medium GlutaMAX supplement (Thermo Fisher Scientific, 36600021) supplemented with 10% FBS (VWR) without antibiotics.
U2OS cells stably expressing a membrane-targeted form of eGFP were generated by transfection with plasmid Lck-GFP (Addgene, 61099 (ref. 37 (link))) and culturing in selection medium (DMEM medium containing 10% FBS, penicillin–streptomycin and 400 μg ml−1 of Geneticin) under conditions of limited dilution to yield single colonies. A clonal cell line with homogenous and moderate expression levels of Lck-eGFP at the plasma membrane was established from a single colony.
All cell lines were tested for mycoplasma (MycoAlert, Lonza) and authenticated by STR profiling (IdentiCell).
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!