Mycobacterial Antigen Preparation and Immune Cell Assays

Mammalian cell culture experiments were completed and Mycobacterium bovis BCG stocks prepared as reported previously (35 (link), 39 (link)). Whole-cell lysates of M. tuberculosis (MtbWL) (NR-14822) were obtained from BEI Resources. Soluble 4-hydroxy-but-2-enyl pyrophosphate (HMBPP; Echelon Bioscience, Salt Lake City, UT) was used for stimulation of γ₉δ₂ T cells as a control in inhibition assays. Interleukin 2 (IL-2; Hoffmann-LaRoche Inc., Basel, Switzerland) was used to expand γ₉δ₂ T cells. Anti-CD3 peridinin chlorophyll protein (PerCP; clone SK7), anti-αβ TCR fluorescein isothiocyanate (FITC; clone B3), anti-γδ TCR phycoerythrin (PE; clone 11F2), anti-CD4 PE-cy7 (clone RPA-T4), anti-CD8 V500 (clone RPA-T8), anti-Ki-67 (clone 35/Ki-67), anti-tumor necrosis factor alpha (anti-TNF-α; MAb11), anti-IFN-γ (B27), and anti-granzyme A (anti-GzmA; CB9) were all from BD Biosciences. Guava ViaCount Flex reagent (Millipore; 4700-0060) was used for accurate counting and discrimination of viable and nonviable cells from antigen-expanded cells. All chemicals for biochemical separations were obtained from Acros Organics (silica gel 60) and Sigma-Aldrich (chloroform and methanol). Alkaline phosphatase (AP) was obtained from Fisher Scientific (BP 80975).

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Xia M., Hesser D.C., De P., Sakala I.G., Spencer C.T., Kirkwood J.S., Abate G., Chatterjee D., Dobos K.M, & Hoft D.F. (2016). A Subset of Protective γ9δ2 T Cells Is Activated by Novel Mycobacterial Glycolipid Components. Infection and Immunity, 84(9), 2449-2462.

Publication 2016

anti-TNF-α; MAb11), anti-IFN-γ (B27), silica gel 60

Alkaline phosphatase Anti cd3 Antigen Assays Cell culture Cell m Cells Chloroform Chlorophyll Clone Discrimination Fitc Fluorescein Guava Gzma Ifn γ Inhibition Isothiocyanate M tuberculosis Mammalian Methanol Mycobacterium bovis bcg Peridinin Phycoerythrin Protein Pyrophosphate Salt Silica gel T cells Tnf α Tuberculosis

Corresponding Organization :

Other organizations : Saint Louis University, Colorado State University

Top 5 similar protocols

Variable analysis

independent variables

Mycobacterium bovis BCG
Whole-cell lysates of Mycobacterium tuberculosis (MtbWL)
4-hydroxy-but-2-enyl pyrophosphate (HMBPP)

dependent variables

Expansion and function of γ9δ2 T cells

control variables

Interleukin 2 (IL-2) for expanding γ9δ2 T cells
Anti-CD3, anti-αβ TCR, anti-γδ TCR, anti-CD4, anti-CD8, anti-Ki-67, anti-TNF-α, anti-IFN-γ, and anti-granzyme A antibodies for flow cytometry analysis
Guava ViaCount Flex reagent for cell counting and viability discrimination
Chemicals (silica gel 60, chloroform, methanol) for biochemical separations
Alkaline phosphatase (AP) for biochemical experiments

positive controls

Stimulation of γ9δ2 T cells with HMBPP

negative controls

Not explicitly mentioned

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