Computational protocol: An approach to the immunophenotypic features of circulating CD4+NKG2D+ T cells in invasive cervical carcinoma

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Protocol publication

[…] Multicolor flow cytometric analysis was performed to detect the frequency and immunophenotypic profile of the CD4+NKG2D+ T cell population. Assay tubes with 100 μL of freshly isolated whole blood from cervical cancer patients or healthy controls was stained with a mixture of the corresponding antibodies for 45 min at 4 °C. After incubation, erythrocytes were lysed using BD FACS Lysing Solution (BD Bioscience, San Jose, CA, USA). The expression of TCR αβ, TCR Vα24Jα18, CD28, CD45RO, CD158b, CD107a, CD161 and HLA-DR was detected on the gate drawn around the CD3+CD4+NKG2D+ lymphocyte population. Firstly, lymphocytes were distinguished from other mononuclear cells using morphological parameters. Then, gate was drawn around the lymphocyte population, which was then analyzed separately using the labeling antibody scheme. Attune® NxT Acoustic Focusing Cytometer (Life Technologies, Carlsbad, CA, USA) was used to acquire twenty thousand events in the lymphocyte region. FACS data were analyzed using FlowJo software, version 8.7 (Tree Star, Inc., Ashland, OR, USA). [...] A commercial magnetic bead-based immunoassay (Bio-Rad Laboratories Inc., Hercules, CA, USA) enable to quantify multiple cytokines in a single well was used to quantify the cytokines in plasmas from cervical cancer patients or control women. Briefly, we designed a kit, which included the detection of pro- and anti-inflammatory cytokines: IL-15, IL-1β, IL-6, IL-17, TNF-α, IFN-γ, IL-2, and IL-10. Thawed samples, standard dilution series, and blanks were pipetted into designated wells; then plate was incubated at RT for 30 min, at 300 rpm shaking to enhance detection assay. After a series of washes to remove unbound protein, a biotinylated detection antibody was added to create a sandwich complex. The final detection complex was formed with the addition of streptavidin-phycoerythrin conjugate. Data were acquired using a Bio-plex system reader and management of results was performed using Bio-Plex Data Pro software. […]

Pipeline specifications

Software tools FlowJo, Bio-Plex Data Pro
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Autoimmune Diseases, Carcinoma, Uterine Cervical Neoplasms, Neoplasms, Cervical Intraepithelial Neoplasia, Sleep Disorders, Circadian Rhythm