Background
Materials and methods
Induction of active EAE
Assessment of spinal cord immune infiltrate by FACS analysis
Antibody | Targets |
---|---|
Anti-CD45.2 | Immune cells (high level) and microglia (intermediate level) |
Anti-CD11b | Granulocytes/neutrophiles, monocytes/macrophages, microglia |
Anti-CD3 | T cells (with anti-CD4 for CD4 T cells; with anti-CD8 for CD8 T cells) |
Anti-B220 | Cells of the B cell lineage |
Anti-CD19 | Cells of the B cell lineage (high level before plasma blast stage) |
Anti-CD44 | Memory/effector T cells |
Anti-CD62L | Naive (CD44−) and central memory (CD44+) T cells |
Anti-Ly6G | Neutrophiles |
Preparation and staining of frozen sections for LCM
Laser capture microdissection and RNA extraction
Taqman arrays
Symbol | ID assay | Gene name |
---|---|---|
Arg1 | Mm00475988_m1 | Arginase 1 |
Ccl2 | Mm00441242_m1 | Chemokine (C-C motif) ligand 2 |
Ccl3 | Mm00441259_g1 | Chemokine (C-C motif) ligand 3 |
Ccl5 | Mm01302427_m1 | Chemokine (C-C motif) ligand 5 |
Ccl7 | Mm00443113_m1 | Chemokine (C-C motif) ligand 7 |
Cxcl9 | Mm00434946_m1 | Chemokine (C-X-C motif) ligand 9 |
Cxcl10 | Mm00445235_m1 | Chemokine (C-X-C motif) ligand 10 |
Cxcl12 | Mm00445553_m1 | Chemokine (C-X-C motif) ligand 12 |
Cxcl16 | Mm00469712_m1 | Chemokine (C-X-C motif) ligand 16 |
Dbi | Mm00833261_g1 | Diazepam binding inhibitor |
Gfap | Mm01253033_m1 | Glial fibrillary acidic protein |
Il15 | Mm00434210_m1 | Interleukin 15 |
Il15ra | Mm04336046_m1 | Interleukin 15 receptor, alpha chain |
Il1b | Mm00434228_m1 | Interleukin 1 beta |
Lcn2 | Mm01324470_m1 | Lipocalin 2 |
Mbp | Mm01266402_m1 | Myelin basic protein |
Serping1 | Mm00437834_m1 | Serpin peptidase inhibitor, clade G, member 1 |
Spp1 | Mm00436767_m1 | Secreted phosphoprotein 1 (osteopontin) |
Sult1a1 | Mm00467072_m1 | Sulfotransferase family 1A, phenol-preferring, member 1 |
Sult1c2 | Mm00471849_m1 | Sulfotransferase family, cytosolic, 1C, member 2 |
Sult1d1 | Mm00502035_m1 | Sulfotransferase family 1D, member 1 |
Sult1e1 | Mm00499178_m1 | Sulfotransferase family 1E, member 1 |
Sult2a1 | Mm04205659_mH | Sulfotransferase family 2A, DHEA-preferring, member 2 |
Sult2b1 | Mm00450550_m1 | Sulfotransferase family, cytosolic, 2B, member 1 |
Sult3a1 | Mm00491057_m1 | Sulfotransferase family 3A, member 1 |
Tgfb1 | Mm01178820_m1 | Transforming growth factor, beta 1 |
Tnf | Mm00443258_m1 | Tumor necrosis factor alpha |
Tnfrsf9 | Mm00441899_m1 | Tumor necrosis factor receptor superfamily, member 9 |
Tnfsf9 | Mm00437155_m1 | Tumor necrosis factor (ligand) superfamily, member 9 |
Tspo | Mm00437828_m1 | Translocator protein |
Ugt1a1 | Mm02603337_m1 | UDP glucuronosyltransferase 1 family, polypeptide A1 |
Ugt2a3 | Mm00472170_m1 | UDP glucuronosyltransferase 2 family, polypeptide A3 |
Ugt2b34 | Mm00655373_m1 | UDP glucuronosyltransferase 2 family, polypeptide B34 |
Ugt2b5 | Mm01623253_s1 | UDP glucuronosyltransferase 2 family, polypeptide B5 |
Ugt3a1 | Mm01703504_mH | UDP glycosyltransferases 3 family, polypeptide A1 |
Ugt8a | Mm00495930_m1 | UDP galactosyltransferase 8A |
Housekeeping genes | ||
Hprt | Mm00446968_m1 | Hypoxanthine guanine phosphoribosyl transferase |
Ppia | Mm02342430_g1 | Peptidylprolyl isomerase A |
Tuba1a | Mm00846967_g1 | Tubulin, alpha 1A |
Gadph | Mm99999915_g1 | Glyceraldehyde-3-phosphate dehydrogenase |
Immunocytochemistry on spinal cord sections
Results and discussion
FACS analysis of EAE infiltrates
RNA is well preserved after the different immunolabeling and LCM steps
TLDA analysis
Mix tissue | LCM-astrocytes | |||||
---|---|---|---|---|---|---|
Ct value | dCt Con | dCt EAE | Fold increase (2−ddCt) | |||
Gene symbol | Detected in all EAE samples at poor to good levels | |||||
Arg1 | 25.1 | 14.3 ± 1.0 | 13.2 ± 1.5 | 2.2 | NS | ¥ |
Ccl2
| 23.3 | 12.0 ± 0.5 | 3.5 ± 0.6 |
349
| * | ¥ |
Ccl3 | 23.9 | 11.1 ± 2.7 | 11.8 ± 4.4 | 0.6 | NS | |
Ccl5
| 18.5 | 12.0 ± 2.5 | 4.4 ± 1.1 |
199
| * | ¥ |
Ccl7
| 23.9 | 12.5 ± 2.1 | 4.6 ± 0.4 |
231
| * | ¥ |
Cxcl9
| 21.3 | 13.0 ± 1.7 | 4.9 ± 0.9 |
270
| * | ¥ |
Cxcl10
| 21.7 | 13.8 ± 1.1 | 6.1 ± 0.6 |
214
| * | ¥ |
Cxcl16
| 21.4 | 9.8 ± 1.9 | 5.0 ± 0.5 |
28
| * | |
Dbi
| 12.8 | 0.7 ± 0.2 | −0.2 ± 0.2 |
1.8
| * | |
Gfap | 24.7 | 9.7 ± 1.2 | 11.9 ± 3.0 | 0.2 | NS | |
Lcn2
| 13.7 | 10.5 ± 2.9 | 2.5 ± 0.4 |
253
| * | |
Mbp | 22.1 | 7.9 ± 1.7 | 7.4 ± 1.5 | 1.4 | NS | |
Serping1
| 16.5 | 11.0 ± 2.5 | 5.4 ± 0.3 |
48
| * | |
Spp1
| 23.2 | 13.1 ± 1.1 | 7.3 ± 1.0 |
54
| * | |
Sult1a1
| 18.1 | 10.5 ± 0.5 | 7.5 ± 0.7 |
7.7
| * | |
Tnf | 33.3 | 10.2 ± 0.8 | 13.9 ± 1.5 | 0.1 | NS | |
Tspo
| 17.6 | 10.7 ± 2.8 | 5.4 ± 1.1 |
31
| * | |
Detected at poor levels in some EAE samples | ||||||
Il1b | 26.2 | Detected only in one EAE sample | ||||
Il15 | 23.4 | Detected only in two EAE samples | ||||
Il15ra | 22.9 | Detected only in two EAE samples | ||||
Tnfrsf9 | 28.8 | Detected only in one EAE sample | ||||
Tnfsf9 | 29.3 | Detected only in one EAE sample | ||||
Detected at very poor levels in all samples | ||||||
Cxcl12 | 24.6 | 13.9 ± 0.8 | 14.6 ± 1.7 | |||
Tgfb1 | 33.6 | 13.9 ± 1.1 | 18.2 ± 0.5 | |||
Ugt8a | 30.1 | 12.7 ± 1.7 | 17.7 ± 0.4 | |||
Undetected in all samples | ||||||
Sult1c2 | 25.0 | Undetected | ||||
Sult1d1 | 22.8 | Undetected | ||||
Sult1e1 | 25.1 | Undetected | ||||
Sult2a1 | 18.3 | Undetected | ||||
Sult2b1 | 23.2 | Undetected | ||||
Sult3a1 | 19.7 | Undetected | ||||
Ugt1a1 | 24.2 | Undetected | ||||
Ugt2a3 | 21.6 | Undetected | ||||
Ugt2b5 | 20.2 | Undetected | ||||
Ugt2b34 | 23.3 | Undetected | ||||
Ugt3a1 | 23.3 | Undetected |