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.
Lamp1 mcherry
Manufactured by Addgene
LAMP1-mCherry is a plasmid that encodes the lysosome-associated membrane protein 1 (LAMP1) fused to the mCherry fluorescent protein. LAMP1 is a widely used marker for lysosomes, and the mCherry tag allows for the visualization of LAMP1-positive organelles.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Sodium hydroxide solution, by Merck Group (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Branched polyethyleneimine pei, by Merck Group (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Sodium alginate, by Merck Group (1 mentions)
Chitosan, by Merck Group (1 mentions)
Rab7 mcherry, by Addgene (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Mrfp gfp lc3b, by Addgene (1 mentions)
Mcherry lc3b, by Addgene (1 mentions)
6 protocols using lamp1 mcherry
1
Polycation-Mediated Gene Delivery System
2
Transfection and Silencing Protocols for Neuronal Proteins
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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).
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).
3
Overexpression and Silencing of STIM1 in Fibroblasts
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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′.
4
Plasmid Transfection and Autophagy Monitoring
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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.
5
Tracking Vesicle Dynamics in 2D and 3D Cultures
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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)).
6
Immunocytochemistry Visualization of P2RX4 and LAMP1
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