LUHMES cryopreservation media 70% Advanced DMEM media 10% FBS 20% DMSO 2.1.2 Biochemicals a) Protein extraction and bicinchoninic acid (BCA) protein assay Extra strong lysis (XSLB) 10mM Tris-HCL (pH 7.5) 0.5% (w/v) sodium dodecyl sulphate (SDS) 0.5% (w/v) sodium deoxycholate 1% (v/v) Triton X-100, 75mM sodium chloride 10mM ethylenediaminetetraacetic acid 2mM sodium orthovanadate 1.25mM sodium fluoride Protease inhibitor cocktail (PIC) for mammalian tissues (1 tablet/ 10ml of XSLB) (Roche, Mannheim, Germany) Phosphatase inhibitor tablets (PhosSTOP™) (1 tablet used/ 10ml XSLB) (Roche, Mannheim, Germany) Bichinoic (BCA) protein assay BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) Bovine serum albumin (BSA) standard Thermo Scientific™Pierce™ Albumin standard (Pierce Endogen, Rockford, IL, USA) b) Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis (SDS-PAGE) 30% Acrylamide/Bis solution (Bio-Rad Laboratories Ltd., Hertfordshire, UK) Ammonium Persulphate (APS) (Cat. No- A3678)
in 5%(v/v) acetic acid diluted in ddH2O. Nitrocellulose membrane (GE Healthcare, Little Chalfont, Bucks, UK) Blocking solutions: Milk-PBST 5% (w/v) Marvel Original Dried Skimmed Milk 0.01% (v/v) Tween 20 in 1x PBS BSA-PBST 5% Albumin bovine fraction V (BSA) (Melford Laboratories Ltd., Chelsworth, Ipswich, UK) 0.01% (v/v) Tween20 in 1x PBS Western Ezier Super Blocking Buffer for Phospho-Specific Antibody Purchased from GenDEPOT to specifically preserve phosphoprotein antigen during blotting (GenDEPOT, Barker, TX, US) Wash buffer 0.01% (v/v) Tween-20 in 1 x PBS Enhanced chemiluminescent substrate (ECL) (EZ-ECL Biological Industries, Israel) Table 2.1. Primary antibodies used for western blotting Primary antibodies used for western blotting in this study. Antibody name, specificity, species, dilution, and source are shown. Abbreviation CST Cell Signaling Technology. Beverly, MA, US Antibody Specificity Species/Type Diluent and dilution Source IGF1Rβ (C-20) C-terminus of the IGF1 receptor β Rabbit polyclonal 5% BSA/PBST 1:50 Santa Cruz (SCBT), Dallas, Texas, US
IRβ C-terminus of the human insulin receptor β Rabbit monoclonal 5% BSA/PBST 1:1000 CST IRS1 Amino acids of IRS-1 human protein. Rabbit polyclonal Western Ezier 1:1000 CST IRS2 IRS-2 human protein Rabbit polyclonal Western Ezier 1:1000 CST Phospho Akt XP (ser473) Human Akt phosphorylated at ser473 site. Rabbit monoclonal Western Ezier 1:1000 CST Akt Akt human protein Rabbit polyclonal Western Ezier 1:1000 CST Phospho-p44/42 MAPK (Erk1/2) MAPK human protein phosphorylated at residue Thr202/Tyr204 Rabbit polyclonal Western Ezier 1:1000 CST p44/42 MAPK (Erk1/2) MAPK human protein Rabbit polyclonal Western Ezier 1:1000 CST β-actin antibody β-actin human protein Mouse monoclonal 5% Milk/PBST 1:10000 Abcam, UK ϒ-H2AX Phosphopeptide containing human Histone H2AX S139 site Rabbit polyclonal Western Ezier 1:1000 R&D systems, MN, USA GFAP GFAP Rabbit polyclonal Western Ezier 1:1000 Abcam, UK Ab7260 Alpha-tubulin Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human alpha Tubulin. Rabbit polyclonal Western Ezier 1:5000 Abcam, UK Ab4074 Neurofilament Light (NFL) NFL Mouse monoclonal Western Ezier 1:1000 Novus ) s , ,
Mounting media Fluoromount Aqueous mounting medium (Sigma F4680) Table 2.5. Primary antibodies used for immunocytochemistry. Antibody reactivity, dilution and source are shown. Antibody Specificity Species/Type Diluent and dilution Source IGF1Rβ (C-20) C-terminus of the IGF receptor β Rabbit polyclonal 3% BSA/PBST 1:25 Santa Cruz (SCBT), Dallas, Texas, US IRβ C-terminus of the human insulin receptor β Rabbit monoclonal 3% BSA/PBST 1:25 Santa Cruz (SCBT), Dallas, Texas, US Vimentin Vimentin Mouse monoclonal 3% BSA/PBST 1:250 Abcam, Cambridge, UK, Ab8978 β3-Tubulin Synthetic peptides from β3-Tubulin Chicken polyclonal 3% BSA/PBST 1:1000 Millipore Lot 2424655 GFAP Full length GFAP Rabbit polyclonal 3% BSA/PBST 1:1000 Abcam, Cambridge, UK, Ab 7260 ALDH1L1 C-terminus of ALDH1L1 Rabbit polyclonal 3% BSA/PBST 1:100 Abcam, Cambridge, UK, Ab 79727 Connexin 43 (Cx43) C-terminal segment of the cytoplasmic domain of Cx43 (amino acids with added lysine) Rabbit polyclonal 3% BSA/PBST 1:100 Sigma C6219 061M4823 CD44 CD44 Mouse monoclonal 3% BSA/PBST 1:100 Dako, Copenhagen, Denmark, Clone DF1485
(NFL) NFL Mouse monoclonal 3% BSA/PBST 1:250 Novus Biologicals NB 300-132 Table 2.6. Secondary antibodies for immunocytochemistry. Antibody reactivity, dilution and source are shown. Antibody Species/Type Diluent and dilution Source Alexa fluor 488 (labelled with green fluorescent dye) Goat anti rabbit IgG (H-L) 3% BSA/PBST 1:1000 Life technologies, Grand Island, NY, USA Alexa fluor 568 (labelled with orange-red fluorescent dye) Goat anti mouse IgG (H-L) 3% BSA/PBST 1:1000 Life technologies, Grand Island, NY, USA Alex fluor 488 (labelled with green fluorescent dye) Goat anti chicken IgG (H-L) 3% BSA/PBST 1:1000 Life technologies, Grand Island, NY, USA 2.1.8 CellRox CellROX® Green Reagent (C10444, Life technologies, Oregon, USA Ref C10444) 2.1.9 Lactate Assay Lactate Assay Kit, including lactate standards (KA0833, Abnova). 2.1.10 Complex I Assay Complex I enzyme activity microplate assay kit (Abcam, ab109721, Cambridge, UK)
GFP LUHMES were co-cultured with DMSO or Wortmannin pretreated astrocytes for 24, 48 and 72 hours. Representative images show GFP LUHMES co-cultured with DMSO and Wortmannin pretreated astrocytes. Bar charts represent the quantification of the neurite outgrowth at each time point. Data represents mean ± SEM (n=3, 3 replicates/experiment, 5 fields counted per coverslip (represents 1 replicate), one way ANOVA with post-hoc analysis **p<0.01 ****p<0.0001).