6-OHDA treatment and cytokine injection
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Animals were kept under pentobarbital sodium anesthesia (50 mg/kg) and placed in a stereotactic instrument .
- 6-OHDA was dissolved in saline containing ascorbic acid (10 μg/μL dissolved in 1% ascorbate-saline), kept on ice (4°C) and protected from light to minimize oxidation, until use.
- The rats were then given uni- or bilateral injections of 6-OHDA.
- Unilateral injection was employed for immunohistochemical analyses, and bilateral injection was used for all other studies.
- For unilateral injection, 5 μL of 6-OHDA was drawn into a Hamilton syringe and then injected into the right side of the striatum, through a hole made on the skull at 1 mm anterior to bregma and 3 mm lateral from the midline.
- The depth of the needle tip was 5 mm from the skull surface.
- The same amount of 6-OHDA was injected into the left side of the striatum for bilateral injection.
- The rate of fluid injection was 1 μL/min.
- The needle was left…
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Animals were kept under pentobarbital sodium anesthesia (50 mg/kg) and placed in a stereotactic instrument .
- 6-OHDA was dissolved in saline containing ascorbic acid (10 μg/μL dissolved in 1% ascorbate-saline), kept on ice (4°C) and protected from light to minimize oxidation, until use.
- The rats were then given uni- or bilateral injections of 6-OHDA.
- Unilateral injection was employed for immunohistochemical analyses, and bilateral injection was used for all other studies.
- For unilateral injection, 5 μL of 6-OHDA was drawn into a Hamilton syringe and then injected into the right side of the striatum, through a hole made on the skull at 1 mm anterior to bregma and 3 mm lateral from the midline.
- The depth of the needle tip was 5 mm from the skull surface.
- The same amount of 6-OHDA was injected into the left side of the striatum for bilateral injection.
- The rate of fluid injection was 1 μL/min.
- The needle was left at the point of injection for an additional 10 min after the injection and then slowly withdrawn.
- Because the bilaterally injected rats could not move well to drink or to eat, they were intraperitoneally injected with electrolyte solution (Solita-T3, Ajinomoto, Tokyo, Japan) twice per day for 1 week.
- A cytokine mixture containing 0.2 mg/mL rat recombinant GM-CSF and 0.2 mg/mL rat recombinant IL-3 was subcutaneously injected from the next day of the 6-OHDA-treatment at a dose of 10 μg/kg body weight (
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DC Preparation and in vitro Challenge
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Enriched DCs were prepared by depleting T and B cells from total splenocytes using purified rat anti-mouse CD3 and anti-mouse CD19 mAbs, then anti-rat IgG magnetic beads , followed by further enrichment using CD11c+ magnetic beads (CD11c+ isolation kit ).
- The enriched CD11c+ DCs were seeded in a 96-well round-bottom tissue culture plate (5×105 cells/well) in RPMI-1640 supplemented with 10% FCS, L-glutamine, Na pyruvate, Hepes, non-essential amino acids and 10−5 M β-ME with GM-CSF (10 ng/ml, ) and challenged with the TLR9 agonist, unmethylated CpG ODN 1668 (CpG; 6 µg/ml).
- The effect of injury on maturation of cDCs was examined at 24 h post-TLR9 activation and pDCs at 40 h post-activation.
- After activation, cells were washed and stained with APC-conjugated anti-mouse CD11c, PE Texas Red-conjugated anti-mouse B220, FITC-conjugated anti-mouse PDCA, PE-conjugated anti-mouse CD80, or CD86 and biotin-conjugated MHC Class-II with…
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Enriched DCs were prepared by depleting T and B cells from total splenocytes using purified rat anti-mouse CD3 and anti-mouse CD19 mAbs, then anti-rat IgG magnetic beads , followed by further enrichment using CD11c+ magnetic beads (CD11c+ isolation kit ).
- The enriched CD11c+ DCs were seeded in a 96-well round-bottom tissue culture plate (5×105 cells/well) in RPMI-1640 supplemented with 10% FCS, L-glutamine, Na pyruvate, Hepes, non-essential amino acids and 10−5 M β-ME with GM-CSF (10 ng/ml, ) and challenged with the TLR9 agonist, unmethylated CpG ODN 1668 (CpG; 6 µg/ml).
- The effect of injury on maturation of cDCs was examined at 24 h post-TLR9 activation and pDCs at 40 h post-activation.
- After activation, cells were washed and stained with APC-conjugated anti-mouse CD11c, PE Texas Red-conjugated anti-mouse B220, FITC-conjugated anti-mouse PDCA, PE-conjugated anti-mouse CD80, or CD86 and biotin-conjugated MHC Class-II with streptavidin PE-Cy7 as secondary.
- cDCs and pDCs were analyzed as CD11chiB220− and CD11clowB220+PDCA+, respectively, by flow cytometry.
- In some cases, cDCs and pDCs were further prepared from the enriched DC population to a higher purity of >98% using a BD FACSAria II cell sorter (cDCs: CD11chighB220−DX5−PDCA−; pDCs: CD11clowB220+PDCA+).
- The sorted cDCs or pDCs were seeded in a 96-well U-bottom plate (2×105 cells/well) and challenged with CpG (6 µg/ml).
- IL-3 (10 ng/ml) was added to the pDC culture to maintain viability.
- Supernatants were harvested 18–20 hr later and tested for levels of IL-6, IL-10, IFN-γ, TNF-α, and IL-12p70 using a Bead Array (CBA ) following the manufacturer’s instructions.
- IL-12p70 and IFN-α (PBL Interferon, , ) production was validated by ELISA assay.
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