RT-qPCR

70–80% confluence astrocyte, microglia or neuronal cultures grown in 6-well culture plates (3.0 × 10⁵ cells) were washed with PBS followed with the isolation of total RNA using the Aurum Total RNA Mini Kit (Bio Rad, Hercules, CA) according to manufacturer’s instruction. Total RNA was dissolved in elution buffer and then, the quantity and purity of mRNA were determined using the NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Complementary DNA (cDNA) synthesis was performed using the iScript cDNA Synthesis Kit as described elsewhere. The cDNA was amplified with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using SsoAdvanced Universal SYBR Green Supermix. The PCR protocol consisted of incubation at 95 °C for 2 min followed by 40 amplification cycles at 95 °C for 5 s and 60 °C for 30 s. Optimum primer pairs for genes of interest and housekeeping gene, glyceraldehydes 3-phosphate dehydrogenase (Gapdh) were designed using Primer Express version 2.0.0 (Table ​(Table1).1). The ΔCt for each RNA sample was calculated by subtracting the mean Ct of housekeeping gene, Gapdh from the mean Ct of the gene of interest and then relative mRNA gene expression was calculated using 2^−ΔΔCt method as described elsewhere. Three independent experiments in triplicate were carried out using three different culture preparations.

Primer sequences for qRT-PCR

F 5′- ACAGACTTTCTCCAACCTCCAG-3’

R 5′- CCTTCTGACACGGATTTGGT-3’

F 5′- CTGGAGAAGGCCATGGTTGC-3’

R 5′- CTCCAGGAAGTGAGAGAGCT-3’

F 5′- CAGTTCTTCAAGGGGTCTGC-3’

R 5′- CAGAATTCGCATCCAAGAGC-3’

F 5′- CCGCTCGCTAAGCTGTTACT-3’

R 5′- CTTTGGTGTTAGAGAGGACAACTTT-3’

F 5′- CAGCCTTTGACCTTATGTCATGG-3’

R 5′- CCTGTGCTGTAGTCGCACT-3’

F 5′-ACCTACAGCATCGGAACCAT- 3’

R 5′-TTGCTAGTAGGGGGTGAAGC-3’

F 5′- CTCAGAGGACAGTGATGTGTTT-3’

R 5′- CGCCTTGCCAGTTATTCTTTG-3’

F 5′- GACTCCTCAGATGAAGTGTTGG-3’

R 5′- GTGCGAAGAAGCCAGAGTAA-3’

F 5′- GTACCGGAGACAGGTGCAGT-3’

R 5′- TTCTCTTCCATCTCACGCATC-3’

F 5′- CCCCATCTCTGCTCACTGTC–3’

R 5′- TTTTTCTGGACCGCATTG-3’

F 5′- GCAACACCCTGGAAGAGATT-3’

R 5′- CTGGGTCTGTTTCCTCACATAG-3’

F 5′- CCCGAATCGTGTCTTTCCTATAA-3’

R 5′- CCTCGATAGAGCTGCAGAATG-3’

F 5′- ACAGTGAAGACCAAGGAAGG-3’

R 5′- CTGGAGGGAGCGTGTATTT-3’

F 5′- AGTAACTGCCGAAGCAAGAA-3’

R 5′- GCACCTCCACATAGCTTACA-3’

F 5′- CCTGGGTTTTCATTGGAGGGTTG-3’

R 5′- GTGGCAGAACTTGAGGAGGTC-3’

F 5′- TGATGGCGCCTTTCTTCTAC-3’

R 5′- CCACCTTGGCCTTCAAAGTA-3’

F 5′- GATACCTACGGCTGGGAAATG-3’

R 5′- GTGATGTCAGCAGGGTAGAAG-3’

F 5′- TCATCGGGTCCTACCTACAA-3’

R 5′- CTTCAGTCACCTGCTCCATAC-3’

F 5′- CGATGCCTTTGGAGGAGTAT-3’

R 5′- GCATAAGGACGGCAGAATAGA-3’

F 5′- GGGCTTCCAGAGCTTTCTATC-3’

R 5′- CTCCAGTTGGCCTACTTCTTC-3’

F 5′- GCCCTTGAGCTCCTCATTG-3’

R 5′- CCCTTACCCACTGGGATGAT-3’

F 5′- TGCTGCCACCTTATTTACTCC-3’

R 5′- AGTGGGGCATGATGACAGTT-3’

F 5′- GCTTCTTCATCAGGGACATCA-3’

R 5′- CTTGAGGGAGAAGTAGGAATG-3’

F 5′- TCCACGGGATTCTAGGAAGA-3’

R 5′- GAGGCAGGAGACTGTGTTAAA-3’

F 5′- TGAGGCTCAGCACAGCAA-3’

R 5′- ATGGGCTTCCAGAATACCG-3’

F 5′- GCCCTCTCTCTCCTCTTGCT-3’

R 5′- GGAGGGTCAGAGCCCATT-3’

F 5′- TGCAGAGGACTCTGAGACAGC-3’

R 5′- GAGTGGTGTCCGAGCCATA-3’

F 5′- CCCCAACCTTCCCAACTC- 3’

R 5′- GGGTTGTAATGGAATGGACAG-3’

F 5′- ATGGGACGGCTGAACAAA-3’

R 5′- GCCATCTCCTCTCTGCAAAT-3’

F 5′- AAACCATCCCAGTGACAAGG-3’

F 5′- GTCCACCTCGAAGCTGTAGG-3’

F 5′- CCTTGATTTTGAGTGGAACAATTT-3’

R 5′- AATGTTGAAAGGACCCTCCAG-3’

F 5′- CCTTGATTTTGAGTGGAACAATTT-3’

R 5′- AATGTTGAAAGGACCCTCCAG- 3’

F 5′- CACCCAGACTCTGTGCTTATT-3’

R 5′- TCAGGTCTCACAGCATCTTTAC-3’

F 5′- CCGCTCGCTAAGCTGTTACT-3’

R 5′- CTTTGGTGTTAGAGAGGACAACTTT-3’

F 5′- GCATCTGTTGGGGAGACAAG-3’

R 5′- TCACCTGGTGGAACATTGTG-3’

F 5′- GGTAATGGAGGGCTTTCTTCTC-3’

R 5′- GCACCGAAGAGAATCAGGTTTA-3’

F 5′- CAAGGCTACGGCCAACAG-3’

R 5′- GTCTTCGTGGGCTTCACTG-3’

F 5′- CCAATCACCGAGAGATGCTTAC-3’

R 5′- CAATGTCCTGGAAGTCGTAGTG-3’

F 5′- CGCTACCAAGATGAAGACACG-3’

R 5′- CAATCACAGGGGGAGAATTG-3’

F 5′- GAACCAACCCTTCGGTGTT-3’

R 5′- TGGATTCTCTGGCTCACACA-3’

F 5′- AGGGATCGACATCCAGAGAG-3’

R 5′- TGCACATTTCCTGTCAAACC-3’

F 5′- CTGCCAACAGTTTTGCTGTG-3’

R 5′- AAATGGCAAACACCCCTCTA-3’

F 5′- CCACAGCATTGAGGAGTTTG-3’

R 5′- ACAGCTCATCATTTGGCTCA-3’

F 5′- CTGCCAGTCCGAAAATGGAAC-3’

R 5′- CTTCATCCACCGGGGCTATC-3’

F 5′- CTGTAGCCCACGTCGTAGC-3’

R 5′- TTGAGATCCATGCCGTTG-3’

F 5′- AGTTGACGGACCCCAAAAG-3’

R 5′- AGCTGGATGCTCTCATCAGG-3’

F 5′- CTCTGAGAACCTCTGAAACGTC-3’

R 5′- GAAACTCAGCCGTCTCTTCTT-3’

F 5′- GAGTCCATACCAGAACCACAC-3’

R 5′- ACAGACAAAGGTCCCACTTG-3’

F 5′- AACACCACCTCAGCCAATAC-3’

R 5′- ACAGCAATGGGAAGAGAACC-3’

F 5′- TGGGACCTCTCCACCAGTT-3’

R 5′- GTGGTGTTGAGGGCTTGG-3’

F 5′- CACTATCCGGAGGTTGCATATC-3’

R 5′- GGAAGACCTTGCTGTTCTCTAC-3’

F 5′- GACGTTCAGCTACTCTGACTTT-3’

R 5′- GTTGGCCTCTTGTGTTCTTTG-3’

F 5′- GAGAGATGGCTCAGTGGTTAAG-3’

R 5′- CACAGGAACAGGGAGCTATTT-3’

F 5′- CTTGAGCTACAGCCATCCTAAC-3’

R 5′- CAGACCACTCTCACAGTTTATCC-3’

F 5′- GTTCCTGACTCCTCTAGCTTTC-3’

R 5′- GCATCTGGTCCAGCCAATA-3’

F 5′- GGGACAGCATTCTGAACTCTAC-3’

R 5′- CTCACTCCTCCCTGATTCTCT-3’

F 5′- AGAAAAGAGAGCTCCATCATGC-3’

R 5′- CGGAATTTCTGGGATTCAGCTTC-3’

F 5′- AACAGCAACTCCCACTCTTC-3’

R 5′- CCTGTTGCTGTAGCCGTATT-3’