Organs were collected and single-cell suspensions were prepared on ice in fluorescence-activated cell sorting buffer (PBS/3% fetal calf serum). All surface stainings were performed for 30 min on ice. Intracellular detection of cytokines and transcription factors were done using the Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences, TNB-0607-KIT), following the protocol provided by the manufacturer. Antibodies used (clone name, dilution factor) were as follows: anti-mouse CD4 BUV395 (GK1.5, 1 : 400), anti-mouse CD8a BUV737 (53-6.7, 1 : 400), anti-mouse Qa-1(b) PE (6A8.6F10.1A6, 1 : 100), anti-mouse CD1d PE (1B1, 1 : 100), anti-mouse PLZF AF647 (R17-809, 1 : 200), anti-mouse CD45.2 BUV737 (104, 1 : 200), anti-mouse RORγt BV786 (Q31-378, 1 : 400), anti-mouse/rat CD44 BV510 (IM7, 1 : 200), anti-mouse IL-4 APC (11B11, 1 : 100), anti-mouse Vβ2 TCR FITC (B20.6, 1 : 100), anti-mouse Vβ3 TCR FITC (KJ25, 1 : 100), anti-mouse Vβ4 TCR FITC (KT4, 1 : 100), anti-mouse Vβ5.1/Vβ5.2 TCR FITC (MR9-4, 1 : 100), anti-mouse Vβ6 TCR FITC (RR4-7, 1 : 100), anti-mouse Vβ7 TCR FITC (TR310, 1 : 100), anti-mouse Vβ8.3 TCR FITC (1B3.3, 1 :100), anti-mouse Vβ10b TCR FITC (B21.5, 1:100), anti-mouse Vβ11 TCR FITC (RR3-15 1 : 100), anti-mouse Vβ12 TCR FITC (MR11-1, 1 : 100), anti-mouse Vβ13 TCR FITC (MR12-3, 1 : 100), and anti-mouse Vβ14 TCR FITC (14-2, 1 : 100) all from BD Bioscience; anti-human HLA-A,B,C PE (W6/32, 1 : 200), anti-mouse I-A/I-E BV510 (M5/114.15.2), anti-mouse H-2Ld/H-2Db PE (28-14-8, 1 : 100), anti-mouse H-2Kb PE (AF6-88.5, 1 : 100), anti-mouse Qa-2 FITC (695H1-9-9, 1 : 100), anti-human/mouse/rat MR1 APC (26.5, 1 : 100), anti-mouse TCRb BV421 (H57-597, 1 : 100), anti-mouse TCR γ/δ PE-Cy7 (GL3, 1 : 100), anti-mouse CD45.1 BV510 (A20, 1 : 200), anti-mouse NK1.1 FITC (PK136, 1 : 100), anti-mouse CD138 PE (281-2, 1 : 200), anti-mouse CD122 PE (TM-β1, 1 : 100), anti-mouse CXCR3 FITC (CXCR3-173, 1 : 100), anti-mouse CD196 (CCR6, 1 : 100) PE (29-2L17), anti-mouse CD25 PE (PC61, 1 : 100), and anti-mouse IL-17A PE (TC11-18H10.1, 1 : 100) all from BioLegend; and anti-mouse/rat Foxp3 PE (FJK-16s, 1 : 200), anti-mouse PLZF AF488 (Mags.21F7), anti-mouse CD69 PE (H1.2F3, 1 : 200), anti-mouse CD19 PerCP-Cy5.5 (eBio1D3, 1 : 200), anti-mouse CD279 (PD-1) PE (J43, 1 : 200), anti-mouse IFNγ PE (XMG1.2, 1 : 100), and anti-mouse Eomes AF488 (Dan11mag, 1 : 100) all from eBioscience. CD1d tertramer loaded with PBS57 (analog of a-galactosylceramide) and MR1 tetramer loaded with 5-OP-RU were provided by the tetramer facility of the US National Institutes of Health. Flow cytometric analysis was performed on LSR Fortessa (BD Biosciences) using FACSDiva software (version 6.1.3, BD Biosciences) and data analysis was done using FlowJo software (Treestar).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.