Two l of the resulting cDNA (5-fold dilution) was subjected to real-time PCR using SYBR Premix Ex lover Taq II (Tli RNase H Plus) master mix (Takara Inc). transformed with the producing pET-21a-mouse S plasmid were resuspended in high-salt buffer (1?M NaCl, 25?mM Tris, pH 7.4, 1?mM EDTA) containing PMSF, subjected to sonication, heated to 100?C for 10?min and centrifuged at 15,000?for 30?min. The supernatant was dialyzed overnight against a 100-fold volume of buffer (25?mM Tris, pH 7.4). The dialyzed sample was ultra-centrifuged at 200,000?for 15?min, the supernatant was applied to a Resource Q column (GE Healthcare) and fractions were eluted with a 0C0.5?M NaCl gradient. Pooled fractions A10 and A11 made up of real, monomeric S as judged by inspection of the SDS gel (Fig.?5A) were used in experiments. LPS in serial dilutions of the protein was measured with a competitive ELISA assay (Elabscience, E-EL-0025) and quantified using an internal LPS standard curve. Quantification of LPS/IFN–induced and S-induced NO production in glia Cells were serum-starved for 24?hours in FBS-free medium Quinagolide hydrochloride (mixed glia and astrocytes) or medium containing 2% FBS (microglia). Subsequently, cells were incubated for 24?hours in DMEM/F12C2% FBS with different concentrations of LPS (Sigma L2880) or recombinant S (as indicated in figures) in presence of 10 ng/ml IFN- (Cell Signaling 5222-SC). NO levels in medium were measured indirectly via quantification of NO-derived nitrite (NO2 -) using the Griess reagent assay78. Briefly, the collected medium was mixed with an equal volume of 1??Griess reagent (Sigma G4410), incubated for 15?min at RT in the dark, and the absorption at 540?nm was measured immediately. Nitrite concentrations were determined using a nitrite standard and normalized to protein content of the same well (measured with the Pierce BCA Rabbit Polyclonal to FZD2 assay). In inhibitor studies, the p38MAPK inhibitor (SB203580), broad-spectrum JNK inhibitor (SP600125) and pan-JAK (Janus kinase) inhibitor were used at 30?M, 20?M and 30?M, respectively. Inhibitors were present from one hour prior to until the end of the 24-h LPS/IFN- treatment. Quantification of inflammatory enzyme and cytokine expression by real-time PCR Cellular RNA was isolated with Trizol reagent, and first strand cDNA was synthesized with the Prime Script RT kit (Takara Inc.) from 500 ng total RNA of each sample. Two l of the producing cDNA (5-fold dilution) was subjected to real-time PCR using SYBR Premix Ex lover Taq II (Tli RNase H Plus) grasp mix (Takara Inc). Forward and reverse PCR primers are outlined in Supplementary Table?1 and were in different exons to avoid amplification of genomic DNA. Melting curve analysis was done to confirm single PCR products. We used the 2 2?Ct method79 to calculate mRNA expression of each gene relative to -actin after initial confirmation that neither loss of PINK1 nor treatment with LPS/IFN- altered the expression of the internal standard -actin (p? ?0.05, t-test). Western blots Main cells were lysed and brain tissue was homogenized with altered RIPA buffer (50?mM Tris-HCl, pH 8.0, 1% Triton X-100, 0.1% SDS, 0.14?M NaCl, 1?mM EDTA, and 1?mM EGTA) containing 1% (v/v) protease inhibitor cocktail (Amresco M250). 20C30?g total proteins in the cleared lysates (supernatants of 10?min, 12,000xcentrifugation) were analyzed by standard Western blot procedures. Anti-GFAP and anti–actin antibodies were used at 4?C overnight, followed by IR-Dye 680RD or IR-Dye 800CW secondary antibodies for 1?hour at room temperature. Bands were visualized using the Odyssey Infrared Imaging System and quantified with ImageJ software. Apoptosis of main neurons co-cultured with mixed astrocytes/microglia Main cortical neurons were isolated from newborn ( 24-hours aged) mice as explained80. Briefly, dissected cortices were washed in HBSS (pH 7.4) containing 1?g/liter D-glucose and digested in 0.25% trypsin at 37?C for 15?min. After addition of 0.014% soybean trypsin inhibitor, tissue was gently triturated Quinagolide hydrochloride in HBSS to generate a suspension of mostly single cells, which was collected by centrifugation and resuspended in neuron growth medium (Neurobasal, 2% B-27 supplement, 0.5 mM L-glutamine, 100 U/ml penicillin, 100?g/ml streptomycin). 1.5??105 neurons/well were added to 15-day old primary mixed glia (3.75??105/well in poly-L-lysine-coated 24-well plates), whose medium was replaced 24?hours earlier with neural growth medium. Half of the medium was changed 24?hours later and subsequently every three days. Six days after plating neurons, the cultures were treated with 1?g/ml LPS and 10 ng/ml IFN- by replacing half of the medium with new LSP/IFN–containing medium, while control cells received 50% new Quinagolide hydrochloride medium only. Twenty-four hours later the cells were fixed with 4% paraformaldehyde and permeated with 0.25% Triton X-100 for immunocytochemistry (ICC). ICC was performed using standard buffers with mouse anti-tubulin-III antibody (neurons) and rabbit anti-active caspase-3 antibody (apoptosis), followed by washing and incubation with species-specific fluorescent secondary antibodies. Confocal images were taken, and the total quantity of neurons (tubulin-III positive cells) and quantity of neurons made up of active caspase-3 (tubulin-III/active caspase-3 double-positive cells) were counted using Image J software.Forty-eight hours later, SH-SY5Y cell viability (survival) was measured with the CCK8 cell counting kit (Dojindo Inc.) according to the manufacturers instructions. supernatant was dialyzed overnight against a 100-fold volume of buffer (25?mM Tris, pH 7.4). The dialyzed sample was ultra-centrifuged at 200,000?for 15?min, the supernatant was applied to a Resource Q column (GE Healthcare) and fractions were eluted with a 0C0.5?M NaCl gradient. Pooled fractions A10 and A11 made up of real, monomeric S as judged by inspection of the SDS gel (Fig.?5A) were used in experiments. LPS in serial dilutions of the protein was measured with a competitive ELISA assay (Elabscience, E-EL-0025) and quantified using an internal LPS standard curve. Quantification of LPS/IFN–induced and S-induced NO production in glia Cells were serum-starved for 24?hours in FBS-free medium (mixed glia and astrocytes) or medium containing 2% FBS (microglia). Subsequently, cells were incubated for 24?hours in DMEM/F12C2% FBS with different concentrations of LPS (Sigma L2880) or recombinant S (as indicated in figures) in presence of 10 ng/ml IFN- (Cell Signaling 5222-SC). NO levels in medium were measured indirectly via quantification of NO-derived nitrite (NO2 -) using the Griess reagent assay78. Briefly, the collected medium was mixed with an equal volume of 1??Griess reagent (Sigma G4410), incubated for 15?min at RT in the dark, and the absorption at 540?nm was measured immediately. Nitrite concentrations were determined using a nitrite standard and normalized to protein content of the same well (measured with the Pierce BCA assay). In inhibitor studies, the p38MAPK inhibitor (SB203580), broad-spectrum JNK inhibitor (SP600125) and pan-JAK (Janus kinase) inhibitor were used at 30?M, 20?M and 30?M, respectively. Inhibitors were present from one hour prior to until the end of the 24-h LPS/IFN- treatment. Quantification of inflammatory enzyme and cytokine expression by real-time PCR Cellular RNA was isolated with Trizol reagent, and first strand cDNA was synthesized with the Prime Script RT kit (Takara Inc.) from 500 ng total RNA of each sample. Two l of the resulting cDNA (5-fold dilution) was subjected to real-time PCR using SYBR Premix Ex Taq II (Tli RNase H Plus) master mix (Takara Inc). Forward and Quinagolide hydrochloride reverse PCR primers are listed in Supplementary Table?1 and were in different exons to avoid amplification of genomic DNA. Melting curve analysis was done to confirm single PCR products. We used the 2 2?Ct method79 to calculate mRNA expression of each gene relative to -actin after initial confirmation that neither loss of PINK1 nor treatment with LPS/IFN- altered Quinagolide hydrochloride the expression of the internal standard -actin (p? ?0.05, t-test). Western blots Primary cells were lysed and brain tissue was homogenized with modified RIPA buffer (50?mM Tris-HCl, pH 8.0, 1% Triton X-100, 0.1% SDS, 0.14?M NaCl, 1?mM EDTA, and 1?mM EGTA) containing 1% (v/v) protease inhibitor cocktail (Amresco M250). 20C30?g total proteins in the cleared lysates (supernatants of 10?min, 12,000xcentrifugation) were analyzed by standard Western blot procedures. Anti-GFAP and anti–actin antibodies were used at 4?C overnight, followed by IR-Dye 680RD or IR-Dye 800CW secondary antibodies for 1?hour at room temperature. Bands were visualized using the Odyssey Infrared Imaging System and quantified with ImageJ software. Apoptosis of primary neurons co-cultured with mixed astrocytes/microglia Primary cortical neurons were isolated from newborn ( 24-hours old) mice as described80. Briefly, dissected cortices were washed in HBSS (pH 7.4) containing 1?g/liter D-glucose and digested in 0.25% trypsin at 37?C for 15?min. After addition of 0.014% soybean trypsin inhibitor, tissue was gently triturated in HBSS to generate a suspension of mostly single cells, which was collected by centrifugation and resuspended in neuron growth medium (Neurobasal, 2% B-27 supplement, 0.5 mM L-glutamine, 100 U/ml penicillin, 100?g/ml streptomycin). 1.5??105 neurons/well were added to 15-day old primary mixed glia (3.75??105/well in poly-L-lysine-coated 24-well plates), whose medium was replaced 24?hours earlier with neural growth medium. Half of the medium was changed 24?hours later and subsequently every three days. Six days after plating neurons, the cultures were treated with 1?g/ml LPS and 10 ng/ml IFN- by replacing half of the medium with fresh LSP/IFN–containing medium, while control cells received 50% fresh medium only. Twenty-four hours later the cells were fixed with 4% paraformaldehyde and permeated with 0.25% Triton X-100 for immunocytochemistry (ICC). ICC was performed using standard buffers with mouse anti-tubulin-III antibody (neurons) and rabbit anti-active caspase-3 antibody (apoptosis), followed by washing and incubation with species-specific fluorescent secondary antibodies. Confocal images were taken, and the total number of neurons (tubulin-III positive cells) and number of neurons containing active caspase-3 (tubulin-III/active caspase-3 double-positive cells) were counted using Image J software to calculate.
Two l of the resulting cDNA (5-fold dilution) was subjected to real-time PCR using SYBR Premix Ex lover Taq II (Tli RNase H Plus) master mix (Takara Inc)