Twenty-four well plates (Corning) were pre-coated overnight at 4 °C with 0.25 µg/mL anti-CD3 (low endotoxin clone UCHT1) and anti-CD28 (low endotoxin clone YTH913.12) antibodies (both from Bio-Rad AbD Serotec Limited, Hercules, CA, USA). The plates were washed three times in PBS (Medicago, Uppsala, Sweden). The CD8+ and CD4+ T cells were cultured in IMDM + 5%FBS (Thermo Fisher Scientific) for 24 h at 37 °C and 5% CO2 at a density of 2 million cells/mL. After culture, a proportion of the cells were processed for flow cytometry and the rest were homogenized and lysed in RLT Plus Buffer (Qiagen) and stored at − 70 °C until extraction. The proportion of live cells (evaluated by LIVE/DEAD™ Fixable Aqua Dead Cell Stain, see details below) was > 68% for the activated CD4+ cells and > 72% for the activated CD8+ cells. The average expression of CD69 after activation in all samples (as determined by flow cytometry) was for CD4+ MS: 5.4% (standard deviation ± 1.8) and HC: 9.5% (± 4.6); CD8+ MS: 15.4% (± 7.2) and HC: 25.6% (± 6.5) (Additional file 1 : Fig. S14).
Anti cd3
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
Anti-CD3 is a laboratory reagent that can be used to detect the presence of CD3, a protein complex found on the surface of T cells. It is commonly used in flow cytometry and other immunoassays to identify and characterize T cell populations.
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Lab products found in correlation
Anti iba1, by Fujifilm (4 mentions)
Anti foxp3, by Thermo Fisher Scientific (2 mentions)
Anti f4 80, by Bio-Rad (2 mentions)
Alexa fluor 405 donkey anti goat, by Abcam (2 mentions)
Anti inos, by Abcam (2 mentions)
Rlt plus buffer, by Qiagen (1 mentions)
Anti neun, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Anti inos, by Novus Biologicals (1 mentions)
Anti cd4, by Abcam (1 mentions)
Anti cd4, by BioXCell (1 mentions)
Anti cd8, by BioXCell (1 mentions)
Anti cd11c, by BioLegend (1 mentions)
Bx43 upright microscope, by Olympus (1 mentions)
Anti mbp, by Merck Group (1 mentions)
Anti smi312, by BioLegend (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti gfap, by Abcam (1 mentions)
Anti cd206, by Bio-Rad (1 mentions)
14 protocols using anti cd3
1
T Cell Activation Protocol
2
Spinal Cord Tissue Immunostaining and Analysis
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Animals were anesthetized with pentobarbital and perfused with ice-cold PBS followed by cold 4% paraformaldehyde. Spinal cords were placed in OCT and deep frozen in dry ice. Serial 10 μm longitudinal sections of spinal cords were prepared, and immunofluorescence stainings were performed as previously described (59 (link)). The following antibodies were used: anti-CD3 (Bio-Rad Laboratories, MCA1477), anti-IBA1 (Fujifilm Wako, 019-19741), anti-iNOS (Novus Biologicals, NBP2-22119), anti-NEUN (MilliporeSigma, MAB377), anti-APC (Ab-7, Calbiochem, OP80), anti-MDA (Abcam, ab6463), anti-GFAP (Dako, Z0334), anti-NG2 (MilliporeSigma, AB5320), anti-MPB (MilliporeSigma, MAB386), and anti-NF M (Chemicon International, AB1987). Goat anti–rabbit Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, A11034), goat anti–mouse Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, A11001), goat anti–rabbit Alexa Fluor 555 (Invitrogen, Thermo Fisher Scientific, A27039), goat anti–rat Alexa Fluor 555 (Invitrogen, Thermo Fisher Scientific, A21434), and goat anti–mouse Alexa Fluor 555 (1:200, Invitrogen, Thermo Fisher Scientific, A28180) were used as secondary antibodies appropriately. Nuclear counterstain was performed using DAPI (Vector Laboratories). Bielschowsky silver impregnation (for axons) and Gold-Black (for myelin) staining were performed as previously described (60 (link)).
3
Immunohistochemistry Staining of Frozen and Paraffin Tissues
4
Immunostaining and Microscopy Analysis of Cryosections
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Cryosections were fixed in acetone for 10 min. Immunostaining and analysis of cryosections were performed as described previously [28 (link)]. The following antibodies were used: anti-CD3 (1:150; Bio-Rad, United States, Hercules), anti-F4/80 (1:100; Bio-Rad), anti-MBP (1:250; Sigma‒Aldrich), and anti-SMI312 (1:250; Biolegend) combined with Alexa Fluor 555-labeled anti-rat IgG and Alexa Fluor 488-labeled anti-mouse IgG secondary antibodies (1:400; Invitrogen, United States, Waltham). Analysis was carried out using a Nikon Eclipse 80i microscope and ImageJ software.
5
Spinal Cord Immune Cell Profiling
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Spinal cord sections were blocked with a blocking solution containing 5% non-fat milk powder, 1% bovine serum albumin and 0.3% Triton-X100 in 0.1 M PBS (all Sigma-Aldrich, USA) for 1 h at room temperature. The following primary antibodies, diluted in the same blocking solution, were then added and incubated at 4 °C overnight: Anti-Iba1 (1:200; Novus Biologicals, USA) for macrophages, anti-iNOS (1:100; Abcam, USA) for M1-macrophages, anti-CD206(1:200; Bio-Rad, Germany) for M2-macrophages, anti-TMEM119 (1:200; Abcam, USA) for microglia, anti-CD3 (1:200; Bio-Rad, Germany) for T-Lymphocytes and anti-GFAP (1:250; Abcam, USA) for astrocytes. Isotype controls with non-specific immunoglobulin at the same concentration were performed to ensure the specificity of the antibodies (data not shown).
As secondary antibodies, Alexa Fluor 405 donkey antigoat (1:400; Abcam, USA), Alexa Fluor 557 donkey antimouse (1:400; R&D Systems, USA) and Alexa Fluor 647 donkey anti-rabbit (1:400; Abcam, USA), diluted in blocking solution without Triton-X100, were used and applied for 1 h at room temperature before covering the sections with mounting medium.
As secondary antibodies, Alexa Fluor 405 donkey antigoat (1:400; Abcam, USA), Alexa Fluor 557 donkey antimouse (1:400; R&D Systems, USA) and Alexa Fluor 647 donkey anti-rabbit (1:400; Abcam, USA), diluted in blocking solution without Triton-X100, were used and applied for 1 h at room temperature before covering the sections with mounting medium.
6
Immunostaining of Immune Cell Infiltrates
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Cryosections were fixed in acetone for 10 min. Immunostaining and analysis of cryosections were performed as described previously [24] .
Briefly, immune cell infiltrates were stained using the following antibodies: anti-CD3 (1:150; Bio-Rad) and anti-F4/80 (1:100; Bio-Rad), combined with the secondary Alexa Fluor 488 or 555-labeled anti-rat IgG antibody (1:400; Invitrogen). Analysis was carried out using a Nikon eclipse 80i microscope and ImageJ software.
Briefly, immune cell infiltrates were stained using the following antibodies: anti-CD3 (1:150; Bio-Rad) and anti-F4/80 (1:100; Bio-Rad), combined with the secondary Alexa Fluor 488 or 555-labeled anti-rat IgG antibody (1:400; Invitrogen). Analysis was carried out using a Nikon eclipse 80i microscope and ImageJ software.
7
Histological and Immunohistochemical Analysis of Gut Samples
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Gut paraffin sections were stained with hematoxylin and eosin (H&E), and analyzed under light microscopy. Gut cryosections (2 μm) were prepared for immunohistochemistry and processed as previously described with the following primary antibodies rat anti-mouse GR-1, rat anti-mouse F4/80 and anti-CD3 (Clone: 145-2C11) (AbD Serotec, Oxford, UK) [23 (link)]. Cytospin preparations of 105 cells were prepared (10 min,600 g) and stained according to May-Gruenwald-Giemsa and analyzed with an Olympus CKX41 light microscope (Shinjuku, Tokyo, Japan) [24 (link)].
8
Histological Analysis of Neuroinflammation
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Tissues were removed from naïve mice or mice with EAE and embedded in paraffin. Tissues were cut into 5-μm-thick sections and stained with H&E to reveal inflammatory infiltrates. For immunohistochemistry, deparaffinized sections were stained with anti-CD3 (AbD Serotec, clone CD3-12), anti-MAC3 (AbD Serotec, clone M3/84), and anti–Iba-1 (Wako Pure Chemical Industries) according to the manufacturer’s protocols. For SALM5 staining, tissues were deparaffinized and rehydrated before Ag retrieval in citrate buffer. Tissues were then stained with different SALM5 antibodies, followed by incubation with Amplification System (K1500, DakoCytomation). After horseradish peroxidase staining, slides were visualized with 3,3′-diaminobenzidine (Sigma-Aldrich).
9
Canine Vaccine IFNγ Response Assay
10
Comprehensive Immunofluorescence Staining Protocol
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For all immunofluorescence stainings, the slides/cells were first blocked with a blocking solution containing 5% non-fat milk, 1% bovine serum albumine, and 0.3% Triton-X100 in 0.1 M PBS for 1 h. The following primary antibodies, diluted in the same blocking solution, were then applied overnight: anti-Nestin (1:400, Millipore) for NPCs, anti-Iba1 (1:1000, Wako) for microglia/macrophages, anti-iNOS (1:500, Abcam) for M1 macrophages, anti-CD206 (1:500, R&D Systems) for M2 macrophages, anti-CD3 (1:100, Serotec) for T lymphocytes, anti-GFAP (1:400, Millipore) for astrocytes, and anti-caspase-3 (1:200, Cell Signaling) for apoptotic cells. Isotype controls with non-specific immunoglobulin at the same concentration were performed to ensure specificity of the antibody stainings (images not shown). Alexa Fluor 568 goat anti-mouse (1:400, Invitrogen), Alexa Fluor 647 goat anti-rabbit (1:400, Invitrogen), and Alexa Fluor 405 donkey anti-goat (1:400, Abcam) diluted in blocking solution without Triton-X100 were used as secondary antibodies and applied for 1 h before covering the slides with mounting medium.
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