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Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cell defense that bridges the innate and adaptive immune systems. ADCC has been found to be a major route of immune protection against both viral infections and cancer. Recently, natural killer (NK) cell-mediated ADCC has been shown as a mechanism of parasite clearance and protection against malaria (Hart et al. J Exp Med 216(6):1280-1290, 2019). NK cells bind to antibodies on the surface of Plasmodium falciparum infected red blood cells (iRBCs) via their Fc receptor, FcγRIIIa (CD16). This interaction induces the release of lytic granules (degranulation) by NK cells, which contain perforin, granzyme B, and granulysin, as well as the secretion of IFNγ. ADCC subsequently limits the growth of Plasmodium falciparum in vitro (Arora et al. Elife:7, e36806, 2018). Because NK cell-mediated ADCC has been shown to be important in malaria parasite clearance and protection, further studies understanding ADCC in malaria are warranted (Hart et al. J Exp Med 216(6):1280-1290, 2019). Therefore, this protocol describes methods to perform an in vitro ADCC functional assay with iRBCs. Specifically, it includes protocols on how to grow and culture iRBCs, how to culture peripheral blood mononuclear cells (PMBCs), and how to do in vitro PBMC assays to measure NK degranulation and IFNγ production as measures of NK ADCC function., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We have described a protocol for performing high-throughput immunofluorescence microscopy on microarrays of yeast cells. This approach employs immunostaining of spheroplasted yeast cells printed as high-density cell microarrays, followed by imaging using automated microscopy. A yeast spheroplast microarray can contain more than 5,000 printed spots, each containing cells from a given yeast strain, and is thus suitable for genome-wide screens focusing on single cell phenotypes, such as systematic localization or co-localization studies or genetic assays for genes affecting probed targets. We demonstrate the use of yeast spheroplast microarrays to probe microtubule and spindle defects across a collection of yeast strains harboring tetracycline-down-regulatable alleles of essential genes.