Multi-Omics Analysis of Immune Cells

Single-cell suspensions of spinal cord, spleen, and inguinal LNs were prepared as previously described [31 (link)]. The following antibodies were used: anti-CD4 PerCP-Cy5.5/APC/eFluor 450/PE-Cy7 (eBioscience, clone RM4-5), anti-CD45.1 FITC (eBioscience, clone A20), anti-CD45.2 PerCP-Cy5.5/APC (eBioscience, clone 104), and anti-CD44 FITC (eBioscience, clone IM7). For intracellular staining, cells were reactivated with culture media (negative control) or 5 μM MOG₃₅₋₅₅ peptide for 7 h with GolgiPlug (BD Biosciences) added for the final 4 h. The following intracellular antibodies were used in accordance with the manufacturer’s protocols: anti-IFNγ eFluor 450 (eBioscience, clone XMG1.2), anti-IL17A Alexa Fluor 647 (eBioscience, clone eBio17B7), anti-GM-CSF PE (BD Biosciences, clone MP1-22E9). A viability dye (Aqua, Life Technologies) was applied to exclude dead cells. Samples were acquired by using an LSRII flow cytometer (BD Biosciences) followed by data analysis using FlowJo version 9.x (Tree Star).

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McWilliams I.L., Rajbhandari R., Nozell S., Benveniste E, & Harrington L.E. (2015). STAT4 controls GM-CSF production by both Th1 and Th17 cells during EAE. Journal of Neuroinflammation, 12, 128.

Publication 2015

anti-CD45.1 FITC anti-CD44 FITC GolgiPlug anti-IFNγ eFluor 450 anti-GM-CSF PE LSRII flow cytometer

Alexa fluor 647 Antibodies Cd44 Cells Clone Culture media Cy5 5 Fitc Gm csf Ifnγ Il17a Inguinal Intracellular Peptide Spinal cord Spleen Tree

Corresponding Organization :

Other organizations : University of Alabama at Birmingham

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Variable analysis

independent variables

Cell treatment (negative control or 5 μM MOG35−55 peptide)

dependent variables

Expression of IFNγ, IL17A, and GM-CSF in CD4+ T cells

control variables

Single-cell suspensions of spinal cord, spleen, and inguinal LNs
Antibodies used for staining: anti-CD4, anti-CD45.1, anti-CD45.2, anti-CD44
Intracellular staining protocol: 7 h reactivation, 4 h GolgiPlug treatment
Flow cytometry acquisition using LSRII and data analysis using FlowJo

controls

Negative control: Culture media
Positive control: 5 μM MOG35−55 peptide

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