Flow Cytometric Analysis of Immune Cells

Cells were isolated from spleens and pancreata as previously described (36 (link)). Lymph nodes were macerated in HBSS (Invitrogen Life Technologies) + 10% FBS (HyClone). Cells were subsequently stained for flow cytometry analysis using the following reagents and reactive antibodies: 7-aminoactinomycin D (7-AAD), B220 (6B2), BCL-6 (K112–91), CCR6 (140706), CD11b (M1/70), CD11c (HL3), CD4 (RM4–5), CD44 (IM7), CD69 (H1.2F3), CD95/FAS (JO2), CD278/ICOS (C398.4A), CXCR3 (CXCR3–173), CXCR5 (2G8), eFluor Fixable Viability Dye, GL7, IgD (11–26c), IgM^a (DS-1), IgM^b (AF6–78), IgM (μ chain specific), NKG2D (cx5), or PD-1 (J43) (BD Biosciences, eBioscience, ThermoFisher Scientific, or Tonbo Biosciences). Biotinylated human insulin (Sigma-Aldrich) (33 (link)) was used to detect insulin-binding specificity. Avidin-fluorochrome conjugates were used to detect biotinylated reagents. Samples were acquired using a BD Biosciences LSR II flow cytometer and data were analyzed using FlowJo software (Tree Star, Inc).

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Bonami R.H., Nyhoff L.E., McNitt D.H., Hulbert C., Felton J.L., Kendall P, & Thomas J.W. (2020). T-B Lymphocyte Interactions Promote Type 1 Diabetes Independently of SLAM-Associated Protein. Journal of immunology (Baltimore, Md. : 1950), 205(12), 3263-3276.

Publication 2020

HBSS NKG2D (cx5), PD-1 (J43) Biotinylated human insulin LSR II flow cytometer

7 aad Antibodies Avidin Bcl 6 Ccr6 Cd11b Cd44 Cells Cxcr3 Cxcr5 Flow cytometry Fluorochrome Hbss Human Icos Insulin Lymph nodes Pancreata Tree

Corresponding Organization : Vanderbilt University Medical Center

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Variable analysis

independent variables

Cells isolated from spleens and pancreata
Lymph nodes macerated in HBSS + 10% FBS

dependent variables

Flow cytometry analysis of cells stained with various reagents and reactive antibodies
Detection of insulin-binding specificity using biotinylated human insulin

control variables

Cells were stained using the following reagents and reactive antibodies: 7-AAD, B220, BCL-6, CCR6, CD11b, CD11c, CD4, CD44, CD69, CD95/FAS, CD278/ICOS, CXCR3, CXCR5, eFluor Fixable Viability Dye, GL7, IgD, IgM, NKG2D, or PD-1

controls

Avidin-fluorochrome conjugates were used to detect biotinylated reagents
Biotinylated human insulin was used as a positive control to detect insulin-binding specificity

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