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Methods in Malaria Research METHODS IN MALARIA RESEARCH Sixth Edition edited by Kirsten Moll, Akira Kaneko, Arthur Scherf and Mats Wahlgren EVIMalaR Glasgow, UK, 2013 MR4/ATCC, Manassas, VA, USA, 2013 copyright © 2013 Kirsten Moll, Akira Kaneko, Arthur Scherf and Mats Wahlgren Coverpicture by Ewert Linder METHODS IN MALARIA RESEARCH 6th edition Welcome to this new edition of Methods in Malaria Research which contains protocols provided by 122 scientists from the global malaria community. The manual is considered a “working document” that, with the help of our readers and users, will continuously grow and evolve as new and improved methods are developed. We, and the contributors hope that the manual will help and assist researchers at all stages of their careers in carrying out frontline research. We express our deep gratitude to all authors who have contributed to Methods in Malaria Research without whose efforts this new edition would not have been possible. The manual was produced with funding from EVIMalaR a European Commission Network of Excellence Funded Project, Glasgow, U.K., MR4/BEI Resources at ATCC, Manassas, USA and Karolinska Institutet, Stockholm, Sweden. The book can be found online at www.evimalar.org, www.mr4.org, www.beiresources.org, and www.ki.se. Other valuable sources for protocols in malaria research are: Malaria Parasites and Other Haemosporidia. P.C.C. Garnham. Blackwell, Oxford, England, 1966; Malaria Methods and Protocols. Series: Methods in Molecular Medicine, vol. 72. D. L. Doolan (Ed.) 2002, Humana Press; Malaria Methods and Protocols. Methods in Molecular Biology, Vol. 923. R. Ménard (Ed.) 2nd ed. 2013. If you have a method you think would be suitable for the next edition or you find some mistakes in the present edition, contact: Kirsten Moll, MTC, Karolinska Institutet, Nobels väg 16, Box 280, SE171 77 Stockholm, Sweden. e-mail: [email protected]. For other comments or questions regarding the protocols, please contact the authors directly. Kirsten Moll Akira Kaneko ArturScherf Mats Wahlgren Stockholm Stockholm Paris Stockholm & Osaka CONTENTS Contributors PARASITES I. Culturing of erythrocytic asexual stages of Plasmodium falciparum and P. vivax A. The candle-jar technique of Trager–Jensen ..........................................................1 B. Establishment of long-term in vitro cultures of Plasmodium falciparum from patient blood ..........................................................................................................3 C. Short-term cultivation of Plasmodium falciparum isolates from patient blood........6 D. Growing Plasmodium falciparum cultures at high parasitemia ..............................7 E. Establishing and growing Plasmodium falciparum patient isolates in vitro with preserved multiplication, invasion and rosetting phenotypes................................ 8 F. Arresting Plasmodium falciparum growth at the trophozoite stage with Aphidicolin .............................................................................................................9 G. In vitro growth of PfEMP1-monovariant parasite culture......................................10 H. Culturing erythrocytic stages of Plasmodium vivax..............................................11 I. In vitro continuous culture of P. vivax by erythroid cells derived from HSCs.......12 II. Freezing and thawing of asexual Plasmodium spp. A. Stockholm sorbitol method .................................................................................17 B. Freezing of patient isolates and strains with glycerolyte .....................................19 C. Thawing of glycerolyte-frozen parasites with NaCl .............................................20 D. Thawing of cryopreserved Plasmodium falciparum using sorbitol ......................21 III. Staining of parasite culture or patient blood and estimation of parasitemia and rosetting rate A. Acridine orange (AO) vital stain of cultures .........................................................22 B. Giemsa staining of thick or thin blood films ....................................................... 23 C. Estimation of the percentage of erythrocytes infected with Plasmodium falciparum in a thin blood film ............................................................................24 D. Estimating the rosetting rate of a parasite culture................................................26 E. Evaluation of parasitemia by lactate dehydrogenase assay................................27 IV. Purification and synchronization of erythrocytic stages A. Enrichment of knob-infected erythrocytes using gelatine sedimentation ............28 B. Sorbitol-synchronization of Plasmodium falciparum-infected erythrocytes .........29 C. Tight synchronisation protocol for in vitro cultures of P. falciparum.....................30 D. Tight synchronization of P. falciparum asexual blood stages for transcriptional analysis................................................................................................................31 E. Enrichment of late-stage infected erythrocytes in 60% Percoll ...........................35 F. Separation of Plasmodium falciparum mature stages in Percoll/sorbitol gradients .............................................................................................................36 G. Obtaining free parasites ......................................................................................38 H. Obtaining semi-intact cells ..................................................................................39 I. Alanine synchronization of Plasmodium falciparum-infected erythrocytes .........40 J. Selection of trophozoites by using magnetic cell sorting (MACS)........................41 K. Isolation of Plasmodium falciparum-infected erythrocytes from the placenta. See: PARASITES section VII:A ..........................................................................69 L. Isolation of viable P. falciparum merozoites by membrane filtration ....................44 V. Micromanipulation cloning of Plasmodium falciparum A. Micromanipulation cloning of parasites................................................................47 B. Micromanipulation cloning of Plasmodium falciparum for single-cell RT-PCR ...48 C. Expansion of Plasmodium falciparum clones .....................................................48 VI. Cytoadhesion and rosetting assays A. Basic cell media ..................................................................................................50 B. Thawing melanoma and other cell lines .............................................................52 C. Freezing of cell lines ...........................................................................................53 D. Cultivation of CHO, COS, HUVEC, melanoma, and L cells ................................54 E. Formaldehyde-fixation of melanoma cells ..........................................................55 F. Binding assays to endothelial and melanoma cells ............................................56 G. Selection of cytoadherent parasites by passage over C32 melanoma, CHO, HUVEC, or other endothelial cells ............................................................58 H. Binding of fluorescent receptors heparin, blood group A, and PECAM-1/CD31 to Plasmodium falciparum-infected erythrocytes ................................................59 I. Adhesion of Plasmodium falciparum-infected erythrocytes to immobilized receptors............................................................................................................. 61 J. Adhesion of Plasmodium falciparum-infected erythrocytes to immobilized hyaluronic acid ....................................................................................................64 K. Binding of Plasmodium falciparum infected erythrocytes to placental tissue sections. See: PARASITES, section VII:B..........................................................71 L. Enrichment of rosetting parasites using Ficoll–Isopaque (Pharmacia) ...............66 M. Reversion of rosettes ..........................................................................................67 VII. Placental malaria A. Isolation of Plasmodium falciparum-infected erythrocytes from the placenta ......69 B. Binding of Plasmodium falciparum-infected erythrocytes to placental tissue sections................................................................................................................71 VIII. Detection of antibodies to the infected erythrocyte surface A. Reversion of rosettes. See: PARASITES, section VI:M.......................................67 B. Agglutination assay using purified trophozoite-infected erythrocytes. Measurement of antibodies to Plasmodium falciparum variant antigens on the surface of infected erythrocytes...........................................................................74 C. Mixed Agglutination Assay: measurement of cross-reactive antibodies to P. falciparum antigens on the surface of infected erythrocytes................................76 D. Serum micro-agglutination of infected erythrocytes.............................................78 E. Flow cytometry detection of surface antigens on fresh, unfixed red blood cells infected with Plasmodium falciparum .................................................................80 F. Analysis by flow cytometry of antibodies to variant surface antigens expressed by P. falciparum-infected erythrocytes.................................................................82 G. Analysis of plasma antibodies to variant surface antigens by flow cytometry......85
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