Improved Plasmodium falciparum dilution cloning through efficient quantification of parasite numbers and c-SNARF detection

Autor(es)

Macedo-Silva, Tatiane ^{[1, 2]} ; Desai, Sanjay A. ^[2] ; Wunderlich, Gerhard ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Biomed Sci, Dept Parasitol, Sao Paulo - Brazil

^[2] NIAID, Lab Malaria & Vector Res, NIH, Rockville, MD 20852 - USA

• Tipo de documento: Artigo Científico
• Fonte: Malaria Journal; v. 20, n. 1 JUN 23 2021.
• Citações Web of Science: 0

Resumo

Background Molecular and genetic studies of blood-stage Plasmodium falciparum parasites require limiting dilution cloning and prolonged cultivation in microplates. The entire process is laborious and subject to errors due to inaccurate dilutions at the onset and failed detection of parasite growth in individual microplate wells. Methods To precisely control the number of parasites dispensed into each microplate well, parasitaemia and total cell counts were determined by flow cytometry using parasite cultures stained with ethidium bromide or SYBR Green I. Microplates were seeded with 0.2 or 0.3 infected cells/well and cultivated with fresh erythrocytes. The c-SNARF fluorescent pH indicator was then used to reliably detect parasite growth. Results Flow cytometry required less time than the traditional approach of estimating parasitaemia and cell numbers by microscopic examination. The resulting dilutions matched predictions from Poisson distribution calculations and yielded clonal lines. Addition of c-SNARF to media permitted rapid detection of parasite growth in microplate wells with high confidence. Conclusion The combined use of flow cytometry for precise dilution and the c-SNARF method for detection of growth improves limiting dilution cloning of P. falciparum. This simple approach saves time, is scalable, and maximizes identification of desired parasite clones. It will facilitate DNA transfection studies and isolation of parasite clones from ex vivo blood samples. (AU)

• Processo FAPESP: 17/24267-7 - Papel de modificadores de cromatina na dinâmica da transcrição de fatores de virulência no parasita da Malária Plasmodium falciparum
• Beneficiário: Gerhard Wunderlich
• Modalidade de apoio: Auxílio à Pesquisa - Regular