Computational protocol: Induction of Protective Immunity to Cryptococcal Infection in Mice by a Heat-Killed, Chitosan-Deficient Strain of Cryptococcus neoformans

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Protocol publication

[…] Briefly, cell surface immunofluorescence staining involved the addition of 0.015 to 0.15 µg of a fluorochrome-conjugated antibody mixture containing antibodies specific to various leukocyte subpopulations to 25 µl of an equal mixture of CSB (defined above) and BD Horizon Brilliant stain buffer (San Jose, CA) in the wells of a 96-well microtiter plate. This staining mixture was then used to resuspend 2.5 × 105 cells in triplicate. Cells were then incubated on ice for 30 min in the dark. This was followed by two washes with CSB, and then cells were fixed at 4°C overnight in 1% paraformaldehyde (PFA). The next day, cells were washed and resuspended in CSB and added to a 96-well microtiter plate for high-throughput flow cytometry sampling using a BD LSRFortessa X-20 (BD Biosciences). BD FACSDiva software was used to process samples on the flow cytometer, and FlowJo (FlowJo, LLC, Ashland, OR) was used for data analysis of the acquired samples to facilitate the identification of leukocyte populations. Thirty thousand cell events were recorded, and dead cells and debris were excluded based upon lower forward scatter (FSC) and side scatter (SSC) signals (i.e., smaller size and granularity). Leukocyte populations were identified by the following markers as previously described: eosinophils (CD45+ CD24+ Siglec F+), neutrophils (CD45+ CD24+ LY6G+), CD11b+ dendritic cells (DCs) (CD45+ CD11b+ CD11c+ CD24+ MHC-II+), CD11b− DCs (CD45+ CD11b− CD11c+ CD24+ MHC-II+), alveolar (CD45+ CD11b− CD11c+ CD24− MHC-II+) and interstitial (CD45+ CD11b+ CD11c+ CD24− MHC-II+) macrophages, Th1 CD4 T cells (CD45+ CD3+ CD4+ CD183+ CD196−), Th2 CD4 T cells (CD45+ CD3+ CD4+ CD183− CD196−), Th17 cells (CD45+ CD3+ CD4+ CD183− CD196+), regulatory T cells (Tregs) (CD45+ CD3+ CD4+ CD25+), CD8 cytotoxic T cells (CD45+ CD3+ CD8+ CD28+), and CD8 T suppressor cells (CD45+ CD3+ CD8+ CD28−) (, , ). The absolute number of each leukocyte population was determined by multiplying the frequency of the leukocyte by the number of cells determined from the single-cell suspensions and then multiplying by 2 to account for the whole lung. […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Mus musculus, Cryptococcus neoformans
Diseases Acquired Immunodeficiency Syndrome, Deficiency Diseases, Infection, Meningoencephalitis, Drug-Related Side Effects and Adverse Reactions
Chemicals Amphotericin B, Flucytosine, Fluconazole