The human neuroblastoma cell line SH-SY5Y, naive or stably expressing APPswe or pcDNA3 (mock), previously described [40 (link)] was exposed to the following drugs for 16–20 h: D6 (50 nM to 5 μM, Elan Pharmaceuticals, San Francisco) in vehicle (methylcellulose/polysorbate 80, Sigma), DAPT (5 μM in DMSO, Sigma), leupeptin (10 μM in H2O, Sigma), PADK (5 μM in DMSO, Bachem), NH4Cl (10 mM in H2O, Sigma), pepstatin A (10 μM in EtOH, Enzo Life sciences), smer28 (50 μM in DMSO, Sigma) and bafilomycin A1 (50 nM in H20, Sigma). Some cells were transfected with GFP-LC3 (Addgene) using Lipofectamine 2000 according to standard protocols. Other cells were infected with lentiviruses (LV) encoding mouse cathepsin B (LV-mCatB-FUGW2) or control (LV-FUGW2) [32 (link)] and polyclonal cell lines stably expressing mCatB (naive SH-SY5Y or APPswe-SH-SY5Y cells) were obtained. For immunocytochemical experiments of C99 expression, COS-7 cells were transfected with C99 using Lipofectamine 2000.
Methylcellulose polysorbate 80
Manufactured by Merck Group
Sourced in United States
Methylcellulose and polysorbate 80 are widely used excipients in various pharmaceutical and laboratory applications. Methylcellulose is a cellulose-derived polymer that acts as a thickening, stabilizing, and suspending agent. Polysorbate 80 is a nonionic surfactant that improves solubility and emulsification. These two components are commonly combined to create a versatile formulation for diverse laboratory and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Pepstatin a, by Enzo Life Sciences (1 mentions)
Smer28, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Nh4cl, by Merck Group (1 mentions)
Lipofectamine 2000, by Addgene (1 mentions)
Gfp lc3, by Addgene (1 mentions)
C57bl 6 mice, by Janvier Labs (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
3 protocols using methylcellulose polysorbate 80
1
SH-SY5Y Neuroblastoma Cell Line Protocols
2
Transgenic AD Mouse Models and Gamma-Secretase Inhibitor Treatment
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3xTgAD (harboring PS1M146V, βAPPswe, and TauP301L transgenes) and non-transgenic (nonTg) mice [37 (link)] were generated from breeding pairs provided by Dr. LaFerla (Irvine, USA). 2xTgAD (PS1wt, βAPPswe and TauP301L) were produced by crossing 3xTgAD with nonTg mice, as described [38 (link)]. For AAV-mediated in vivo delivery, 1-day-old C57BL6 mice (Janvier Labs., France) were injected with 4 µl of AAV virus (5.5 × 1012 vg/ml (viral genomes per ml)) into the left lateral ventricle, as described [21 (link)] and mice were analyzed at 2 months post-AAV delivery. 5-month-old nonTg and 3xTgAD males or 2-month-old AAV-infected mice (males and females) were treated daily for 12 or 30 days with the γ-secretase inhibitor ELND006, referred to as D6 hereafter (30 mg/kg, Elan Pharmaceuticals, San Francisco, USA) [9 , 45 (link)] or with vehicle alone (methylcellulose/polysorbate 80, Sigma) via oral gavage, as described [24 (link)]. All experimental procedures were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and local French legislation.
3
Cellular Models for Alzheimer's Disease
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Human SH-SY5Y neuroblastoma cells (CRL-2266, ATCC) stably expressing pcDNA3.1 (control), wild type human AβPP (APPwt), or human AβPP harboring the double Swedish mutations (APPswe: APPKM670/671NL) constructs were generated as already described [22 (link)]. Polyclonal stable lines were maintained in DMEM containing 10% FBS in the presence of 400 μg geneticin (Gibco).
Mock-transfected or APP695LDN-expressing CHO cells were obtained by stable transfection of pcDNA4 empty vector (Control), wild type hAPP695 cDNA or harboring the London mutation (APPLDN: APPV642I) and subcloned in pcDNA4 vector. Single clones were maintained in DMEM containing 10% FBS, sodium hypoxanthine-thymidine supplement, and 300 μM proline as already described [23 (link)].
Cells were treated overnight (20 h) with β- or γ-secretase inhibitors. γ-secretase inhibitor ELND006 was used at 5 μM final concentration and vehicle (methylcellulose/polysorbate 80; Sigma-Aldrich) was used as control [24, 25 (link)]. β-secretase inhibitor (Eli Lilly’s inhibitor LY288672 [26 (link)], synthesized by Elan Pharmaceutical) was used at 30 μM final concentration prepared in DMSO. Vehicle was used as control since no difference was observed as compared to cells treated with DMSO.
Mock-transfected or APP695LDN-expressing CHO cells were obtained by stable transfection of pcDNA4 empty vector (Control), wild type hAPP695 cDNA or harboring the London mutation (APPLDN: APPV642I) and subcloned in pcDNA4 vector. Single clones were maintained in DMEM containing 10% FBS, sodium hypoxanthine-thymidine supplement, and 300 μM proline as already described [23 (link)].
Cells were treated overnight (20 h) with β- or γ-secretase inhibitors. γ-secretase inhibitor ELND006 was used at 5 μM final concentration and vehicle (methylcellulose/polysorbate 80; Sigma-Aldrich) was used as control [24, 25 (link)]. β-secretase inhibitor (Eli Lilly’s inhibitor LY288672 [26 (link)], synthesized by Elan Pharmaceutical) was used at 30 μM final concentration prepared in DMSO. Vehicle was used as control since no difference was observed as compared to cells treated with DMSO.
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