The largest database of trusted experimental protocols

6 protocols using lamp1 mcherry

1

Polycation-Mediated Gene Delivery System

Check if the same lab product or an alternative is used in the 5 most similar protocols
Sodium alginate (Alg, low viscosity), chitosan (Chi, 75%–85% deacetylated, MW 50–190 kDa), branched polyethyleneimine (PEI, MW 25 kDa), sodium hydroxide solution (NaOH, 1N), acetic acid (ACS reagent), and Amicon ultra-centrifugal filter (4mL, 100 kDa cutoff) were purchased from Sigma-Aldrich. Calcium chloride (dihydrate) were purchased from Mallinckrodt Chemicals. Propidium Iodide (PI, Invitrogen), hydrochloric acid solution (HCl, 1N), and standard RC dialysis membrane (Spectrum® Laboratories, MWCO 3.5 kDa) were purchased through Thermo Fisher Scientific. D-Luciferin was purchased from Xenogen. The plasmid DNA (pDNA), pEGFP-N1, encoding enhanced green fluorescence protein (EGFP), were obtained from Clontech (#6085-1); pcDNA3-luciferase (#18964) encoding Firefly Luciferase gene and pDNA encoding Lysosomal-Associated Membrane Protein 1 (LAMP1)-mCherry (#45147) were purchased from Addgene.
+ Open protocol
+ Expand
2

Transfection and Silencing Protocols for Neuronal Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
All cultures were transfected with Lipofectamine 2000 following the manufacturer’s protocol (Life Technologies). Strep-FLAG NEGR1 was described in Pischedda and Piccoli (2015) (link), hFGFR2-GFP in Schuller et al. (2008) (link), and FGFR2-myc in Mansukhani et al. (2000) (link). RAB5A-mCherry (Addgene 27679), RAB7-mCherry (Addgene 55127) and LAMP1-mcherry (Addgene 45147) were purchased. The siRNA-resistant Strep-FLAG NEGR1 construct was generated by site-directed mutagenesis to introduce three silent mutations within the siRNA target sequence using the forward primer: 5′-GCCGTGGACAACATGCTCGTCAGGAAAGGTGACACAGCG-3′.
Lentiviral vectors expressing a siRNA cassette and GFP reporter were originally described in Wiznerowicz and Trono (2003) (link). The mouse Negr1 and mouse Fgfr2 silencing lentiviral vectors used in biochemical assays (siNegr1 and siFGFR2) and in utero electroporation experiments (siNegr1) are described in Pischedda and Piccoli (2015) (link). The FGFR2 silencing vectors used in in utero electroporation experiments (siRNA 2A: 5′-GCACACACTTACAGAGCACAA-3′ targeting mouse Fgfr2 3′UTR and siRNA 2B: 5′-CCTCTCTACGTCATAGTTGAA-3′ targeting mouse Fgfr2 exon 11) were originally described in Tarkkonen et al. (2012) (link). The silencing cassettes, including a U6 promoter, and silencing sequences 2A and 2B were also cloned into a pDsRed-expression vector (Clontech).
+ Open protocol
+ Expand
3

Overexpression and Silencing of STIM1 in Fibroblasts

Check if the same lab product or an alternative is used in the 5 most similar protocols
LAMP1-mCherry was obtained from Addgene (#45147). STIM1-mcherry was obtained from R. Lewis52 (link). Fibroblasts were transfected with the Neon transfection system (Invitrogen), according to the manufacturer’s instructions, using the following parameters: 1350 V, 30 ms, and one pulse. For overexpression studies, we used 0.5 µg DNA per 50 × 103 cells and the analysis was performed 24 h after transfection. For silencing studies, 50 × 103 cells were transfected with 1 pmol siRNA (Invitrogen) and analyzed 48 h later. The sequence of siRNA targeting STIM1 was 5′-GCAAGGAUGUUAUAUUUGATT-3′, that targeting clathrin heavy chain, 5′-CAUUGUCUGUGAUCGGUUUTT-3′, that targeting TFEB, 5′-CAACCUAAUUGAGAGAAGATT-3′, and that targeting calcineurin, 5′-GGGUUUGGAUAGGAUCAAUTT-3′.
+ Open protocol
+ Expand
4

Plasmid Transfection and Autophagy Monitoring

Check if the same lab product or an alternative is used in the 5 most similar protocols
The cDNA plasmids mCherry-LC3B (Cat# 40827), mRFP-GFP-LC3B (Cat# 21074), and Lamp1-mCherry (Cat# 45147) were purchased from Addgene. All plasmids were validated by sequencing. For transient transfections, the cells were plated at 60%–70% confluence. After 12 h, the cDNAs were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Cat# 11668019) using the forward transfection protocol provided by the manufacturer. The siRNA target sequences are as follows: non-targeting control siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′; human ATG5_siRNA, 5′-GCCUGUAUGUACUGCUUUA-3′; mouse Atg5_siRNA, 5′-CUCUCUAUCAGGAUGAUTT-3′; all the siRNAs have been validated by western blot showing > 80% knockdown of the target.
+ Open protocol
+ Expand
5

Tracking Vesicle Dynamics in 2D and 3D Cultures

Check if the same lab product or an alternative is used in the 5 most similar protocols
Vesicle-associated membrane protein 3 (VAMP3)-enhanced green fluorescent protein (EGFP) (plasmid 42310; Addgene) and LAMP1-mCherry (plasmid 45147; Addgene) were transfected into HT1080 cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to product specifications. Six hours after transfection, the transfection reagent–containing medium was replaced with fresh medium. After 24 h, cells were trypsinized and plated on glass-bottom 24-well plates or embedded inside 3D collagen I gels as mentioned above. The next day (48 h after transfection), cells were imaged using a Nikon A1 confocal at 1 or 2 frames per second depending on the size of the scanned region. VAMP3-EGFP and LAMP1-mCherry dynamics were analyzed using the particle-tracking module in the U-Track software package (24 (link), 26 (link)).
+ Open protocol
+ Expand
6

Immunocytochemistry Visualization of P2RX4 and LAMP1

Check if the same lab product or an alternative is used in the 5 most similar protocols
For immunocytochemistry, cells grown on coverslips and fixed in 4% paraformaldehyde in Dulbecco’s phosphate-buffered saline (DPBS) for 15 min were permeabilized with 0.2% triton X-100 in DPBS for 15 min and blocked in 5% goat serum in DPBS for 30 min before staining with indicated primary antibodies (Table S1) followed by appropriate AlexaFluor488- or AlexaFluor594–coupled secondary antibodies. Nuclei were labeled with 5 mg/ml Hoechst 33342 and coverslips were mounted with Prolong Gold Antifade mounting medium. Images were acquired on LSM700 with a 63X Carl Zeiss oil-objective and the Zeiss Zen software. For P2RX4 and LAMP1 colocalization, cells were transfected with the P2X4-pHluorin123 (a gift from Baljit Khakh, Addgene plasmid # 52926) [29 (link)] and LAMP1-mCherry (a gift from Michael Davidson Addgene plasmid # 55073) plasmids for 24 h, after which the medium was replaced with Live Cell Imaging Solution supplemented with 20 mM HEPES and the images were acquired as above.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!