Name: in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry
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Reconhecemos financiar o apoio do Conselho de Pesquisa Nacional de Saúde e Medicina (conceder APP605524, 490037 e 1047082), o Australian Research Council (conceder DP12010061), a Estratégia Nacional de Infra-estrutura Collaborative Research, da Austrália e do fundo de investimento de Educação do Departamento de Inovação, Indústria , Ciência e Investigação. PML é um receptor de um prêmio de Pós-Graduação da Austrália.

Todos os procedimentos foram conduzidos de acordo com as políticas da Universidade Macquarie e conformado com o Conselho Nacional de Saúde e Pesquisa Médica (NHMRC) Código australiano de prática. O trabalho foi realizado sob as Ética Nenhum Acordo ARA 2012/017 aprovados e obtido da Comissão de Ética Animal da Universidade de Macquarie. Todas as experiências foram realizadas em ratinhos SJL / J a menos que indicado de outra forma. 1. Ratos e Experimental Malaria Infection <ol><li> Camundongos com temperatura controlada (21 ° C) com um ciclo claro-escuro de 12:12 hr casa. </li><li> Descongelar uma aliquota de criopreservado P. chabaudi Adami DS com 5% de hemácias parasitadas (PRBCs) e injetar 200 mL na cavidade intra-peritoneal de camundongos C57BL / 6 camundongo doador. </li><li> Monitorar doador rato parasitemia a cada 1-2 dias, utilizando a citometria de fluxo, conforme descrito na Seção 3-4 do presente protocolo. Uma vez que os doadores atinge 5-15% parasitemia, coleta de sangue por punção cardíaca, como segue: <ol><li>Aspirar 100 ul de solução de heparina (300 U / ml de heparina em solução de Ringer rato (MTR) (NaCl 154 mM, KCl 5,6 mM, MgCl2 1 mM, 2,2 mM de CaCl2, 20 mM de HEPES, 10 mM de glucose, 30 U / ml heparina, pH 7,4, filtro de 0,22 ^ M esterilizado)) numa seringa de 1 ml com uma agulha G 26. </li><li> Anestesiar o mouse por inalação com 5% de isoflurano, confirmando a profundidade desejada da anestesia, pela ausência de resposta de retirada pedal e dos reflexos da córnea. </li><li> Coloque rato sobre o seu lado e perpendicularmente agulha de inserção, logo abaixo do cotovelo, através das costelas, e no coração. Puxe o êmbolo da seringa lenta e gire a agulha até 0,5-1 ml de sangue é obtido. </li><li> Execute eutanásia humanitária por deslocamento cervical sob anestesia. </li></ol></li><li> Calcular as concentrado de hemácias por volume de sangue do dador assumindo uma contagem de sangue de 9 x 10 6 de RBC / ul (ou seja, se o doador estava em 10% quando a parasitemia sacrificados, o sangue conterá 9 x 10 5CH / ul). Dilui-se sangue parasitadas em solução salina de Krebs (NaCl 126 mM, KCl 2,5 mM, NaHCO 3 25, 1,2 mM de NaH 2 PO 4, 1,2 mM MgCl2, 2,5 mM de CaCl2, 0,2% de glucose, pH 7,2, filtro de 0,22 ^ M esterilizado) tamponada de modo que a concentração é de 1 x 10 4 concentrado de hemácias por 200 ul e 200 ul injectar na cavidade intraperitoneal de ratinhos necessárias para a marcação de RBC e experimento transfusão. </li><li> Monitorar parasitemia a cada 1-2 dias, utilizando a citometria de fluxo, conforme descrito nas secções 3-4 deste protocolo. </li></ol> 2. A marcação de hemácias e Transfusão <ol><li> Recolha de sangue heparinizado a partir de murganhos, por punção cardíaca, tal como descrito no passo 1.3, e colocar em gelo. Manter sangue a 4 ° C em todos os momentos. Colete aproximadamente 200 sangue ul por mouse para ser injetado. Se necessário, dividida em duas amostras de sangue e tratar uma amostra de como desejar. NOTA: Desta forma, a invasão do samp tratadale pode ser comparado com um controlo, uma amostra não tratada (ver Figura 1 para esquemática). </li><li> Prepara-se uma solução de cada etiqueta fluorescente RBC 2x. <ol><li> Dilui-se uma solução estoque a 2 mg / ml de Atto 633-N-hidroxisuccinimida (NHS Atto-633) em MTR, de modo que a concentração é de 20 ug / ml. </li><li> Dilui-se uma solução estoque de 25 mg / ml de biotina-N-hidroxissuccinimida (biotina-NHS) em MTR, de modo que a concentração é de 250 ug / ml. </li></ol></li><li> Dividir as amostras de sangue em dois tubos e adicionar um volume igual de 2x solução Atto 633-NHS para um tubo, e um volume igual de 2x a biotina-NHS para outro tubo. Misturar imediatamente. Incubar sangue a 4 ° C durante 1 h, com agitação lenta constante, ou, alternativamente, misturar cada amostra de 10 - 15 min. </li><li> Lavar 3 vezes sangue marcadas com MTRC (MTR + 0,5% albumina de soro bovino (BSA), filtro de 0,22 ^ M esterilizado) como se segue: <ol><li> Adicionar pelo menos dois volumes de MTRC, centrifugar a 750 xg durante 5 minutos, remover o sobrenadante, e resuspend o sedimento em, pelo menos, 2 volumes de MTRC. Repita mais duas vezes. </li><li> Após a lavagem final, centrifugar a 750 xg durante 5 min e combinar o sangue sedimentadas em diferentes combinações (ou seja, sem tratamento Atto 633 / Biotina tratada e não tratada Biotina / tratada Atto 633). Adicionar MTR para fazer até um volume de 200 mL por rato para ser injetado. </li></ol></li><li> Injetar sangue marcado intravascular em camundongos infectados roedor malária ou controles não infectados como se segue: <ol><li> Execute transfusão a 2 - 15% parasitemia no auge de parasita esquizogonia. NOTA: isto ocorre aproximadamente a meio do ciclo escuro (ou seja, entre 12-2 da manhã de um ciclo de luz normal, ou 12-2 pm em um ciclo de luz inverso). </li><li> Camundongos morna usando uma lâmpada de calor por 5-10 minutos para aumentar o fluxo sanguíneo, mas tomar precauções para não superaquecer os ratos. Coloque os ratos em um dispositivo de retenção e intravascular injetar 200 mL de sangue marcado (aproximadamente 1 - 2 x 10 9 RBCs) através da veia da cauda. </li></ol></li><li> Coletar amostras de sangue em diferentes momentos após a injeção, conforme descrito na Seção 3. NOTA: As taxas de invasão pode ser quantificada inferior a 10 minutos após a injecção, dependendo da parasitemia do rato destinatário. </li></ol> 3. Recolha de amostras de sangue e Preparação para citometria de fluxo <ol><li> Preparar 50 ml de solução de coloração por amostra, e um pouco mais, no dia da experiência como se segue: <ol><li> Descongelar uma aliquota de 6 JC-1 mM armazenado a -20 ° C em dimetil-sulfóxido (DMSO). </li><li> Quente 50 ul de MTRC por amostra, e um pouco mais, a 37 ° C. </li><li> Embora a vórtex continuamente MTRC pré-aquecido, adicionar JC-1, de modo que a concentração final é de 12 uM. Isto é feito para reduzir a formação de JC-1 agregados; se agregados que formam, fazer tubo não centrífuga, pois isso vai evitar manchas. NOTA: Uma pequena quantidade de agregação não afetará o resultado. </li><li> Adicionar anti-CD45 da APC eFluor 780 e anti-CD71 PerCP eFluor 710 de modo a que a concentração final é de 1 ug / ml. Se o experimento RBC marcado, adicionar estreptavidina PE-Cy7 de modo que a concentração final é de 1 ug / ml. </li></ol></li><li> Realizar cauda sangramento por amputação da ponta da cauda ou a laceração do vaso sanguíneo dentro da cauda. Coloque uma gota de sangue da cauda para um pequeno pesar barco ou lâmina de vidro. Após a coleta de sangue, garantir que o sangramento parou. Pipeta 3 ul de sangue da cauda em 50 ul de solução de coloração pré-aquecido a 37 ° C e incubar as amostras a 37 ° C durante 20 min. </li><li> Descongelar uma aliquota de 4 mM de Hoechst 33342 armazenado a -20 ° C em água destilada (se usando um laser 355 nm) ou Hoechst 34580 2 mM armazenado a -20 ° C em água destilada (se usando um laser de 405 nm). Prepare 500 mL por amostra, além de extra, de uma solução de 4 mm ou 2 M de Hoechst em MTRC, respectivamente. </li><li> Adicionar 500 uL de 4 uM Hoechst33342 ou 2 uM Hoechst 34580 para as amostras e incuba-se à temperatura ambiente durante 20 min. </li><li> Centrifugar células a 750 xg durante 3 min a 4 ° C, o sobrenadante de descarte, adicionar 700 ul MTRC e analisar por citometria de fluxo. </li></ol> 4. Citometria de Fluxo <ol><li> Analisar amostras usando um laser 355/488/633 nm ou 405/488/633 instrumento a laser nm. </li><li> Amostras de filtrar através de um filtro de células 35 mM imediatamente antes da análise. Enxágüe filtro de células com água destilada entre amostras e re-uso. </li><li> Grave eventos suficientes para que a menor população contém, pelo menos, 500 eventos (geralmente 1.000.000 - 10.000.000 Eventos totais por amostra). </li><li> Excite Hoechst 33342 usando um laser de 355 nm e detectar eventos através de um filtro 460/50. Excitar a Hoechst 34580 usando um laser de 405 nm e detectar eventos através de um filtro 460/50. Excitar JC-1, anti-CD71 PerCP eFluor 710 e PE-estreptavidina Cy7 utilizando um laser de 488 nm e detectar eventos através de um 530/40, 692/40, e filtrar 750LP respectivoly. Excite Atto 633 e anti-CD45 APC eFluor 780 usando um laser de 633 nm e detectar eventos através de um filtro de 670/30 ou 750LP respectivamente. </li><li> Realizar análise e compensação usando citometria de fluxo apropriado software de análise da seguinte forma: <ol><li> Selecione as células inteiras e não incluem ruído, resíduos, e plaquetas com base em FSC / propriedades SSC (Figura 2A). Selecione células individuais com base em qualquer largura de pulso de disparo (Figura 2B) ou usando a área do pico do FSC e à altura (Figura 2C). </li><li> Escolher a eritrócitos maduros, e excluir progenitores e leucócitos de RBC, com base na fluorescência negativa nas PerCP eFluor 710 e 780 APC eFluor canais (Figura 2D). </li><li> Se a realização do experimento RBC marcado, selecione cada população de hemácias marcadas com base em PE-Cy7 e Atto 633 fluorescência e analisá-las separadamente (Figura 3A). </li><li> Selecione concentrado de hemácias com base em fluorescência positiva para ambos JC-1 e Hoechst ( <strong&gt; Figura 2E-H). </li></ol></li></ol> 5. cálculos e estatísticas <ol><li> Calcular a parasitemia por divisão do número de concentrado de hemácias pelo número total de glóbulos vermelhos (ou seja, o número de eventos em Q2 portão dividido pelo número de eventos no portão G4). Ao realizar o ensaio de invasão de parasitemia as duas populações marcados podem ser calculadas dividindo o número de concentrado de hemácias marcadas pelo número de glóbulos vermelhos marcados em que a população (isto é, o número de eventos que são tanto em portas de L1 e Q2, dividido pelo número de eventos no portão L1). NOTA: Rotulado parasitemia varia consideravelmente dependendo de fatores como a parasitemia endógeno e número de merozoites circulantes. Por esta razão, é útil para relatar a proporção de parasitemia, que é calculado como a parasitemia da população marcada tratados dividido pelo parasitemia da população de controlo marcado em cada ratinho individual. Para corrigir possíveis efeitos da tintura, relatório resulta umaé uma relação média parasitemia nas duas combinações de corantes. </li><li> Determinar a significância estatística dos índices usando o teste t uma amostra com 1 como a média hipotética. </li></ol>

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Descrevemos um método para a medição de ambos parasitemia e merozóitos de invasão in vivo amostras. Em relação à medida da parasitemia, este método oferece uma vantagem sobre os métodos anteriores em que 10-13 HJ-GVs podem ser distinguidas a partir de concentrado de hemácias, reduzindo assim o número de eventos positivos falsos. Enquanto HJ-hemácias são geralmente rara em humanos, alguns estudos relatam altos níveis em ratos 15,16 fazendo a distinção entre essas células e...

The clinical symptoms associated with malaria occur during the Plasmodium parasite&#8217;s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs. 
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia. 
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

<table><tbody><tr><td>bisBenzimide H 33342 trihydrochloride</td><td>Sigma-Aldrich</td><td>B2261</td><td>Hoechst 33342. Store a 4 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>Hoechst 34580</td><td>Sigma-Aldrich</td><td>63493</td><td>Store a 2 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>JC-1 Dye</td><td>Life Technologies</td><td>T-3168</td><td>Store small aliquots of 6 mM stock solution at -20 &amp;deg;C in DMSO</td></tr><tr><td>Anti-Mouse CD45 APC-eFluor 780</td><td>eBioscience</td><td>47-0451-80</td><td>Clone 30-F11</td></tr><tr><td>Anti-Mouse CD71 PerCP-eFluor 710</td><td>eBioscience</td><td>46-0711-80</td><td>Clone R17217</td></tr><tr><td>Atto 633 NHS ester</td><td>Sigma-Aldrich</td><td>1464</td><td>Atto 633-NHS. Store a 2 mg/ml stock solution at -20 &amp;deg;C in DMF</td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin</td><td>Thermo Fisher Scientific</td><td>21335</td><td>Biotin-NHS. Store a 25 mg/ml stock solution at -20 &amp;deg;C in DMF</td></tr><tr><td>Streptavidin PE-Cyanine7</td><td>eBioscience</td><td>25-4317-82</td><td>Streptavidin PE-Cy7</td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H478</td><td></td></tr><tr><td>35 &amp;micro;M filter cap tubes</td><td>Becton Dickinson</td><td>352235</td><td></td></tr><tr><td>Flow cytometer: BD LSRFortessa</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: BD FACSAria II</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: BD Influx</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: CyAn ADP Analyzer</td><td>Beckman Coulter</td><td></td><td></td></tr></tbody></table>

in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

Medição da parasitemia. Para a medição da parasitemia, células sanguíneas devem primeiro ser seleccionado, e o ruído, detritos e plaquetas excluídos, com base no FSC / SSC propriedades (Figura 2A). Dependendo do citómetro usado, células individuais devem, então, ser seleccionado com base em qualquer largura de impulsos de disparo (Figura 2B), ou FSC altura de pico com razão de área (Figura 2C). Restante eventos deve ser composto de...

Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Os invade parasita da malária e repetições dentro das células vermelhas do sangue. A avaliação precisa da invasão merozoite e parasitemia é fundamental para avaliar o curso da infecção da malária. Aqui nós descrevemos um protocolo de citometria de fluxo com base para a medição desses parâmetros em um modelo de rato da malária.

A avaliação in vivo de Rodent Plasmodium parasitemia e Merozoito Invasion por citometria de fluxo

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

Research

JoVE Journal

Immunology and Infection

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

9.9K visualizações.  Macquarie University.  Os invade parasita da malária e repetições dentro das células vermelhas do sangue. A avaliação precisa da invasão merozoite e parasitemia é fundamental para avaliar o curso da infecção da malária. Aqui nós descrevemos um protocolo de citometria de fluxo com base para a medição desses parâmetros em um modelo de rato da malária. 

Vídeo: In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Protocolo para produção de uma Cruz genética do parasita da malária de roedores

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Imunologia e Infecção

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Microscopia intravital da Microcirculação cérebro de camundongo usando uma janela fechada Cranial

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neurociência

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Microscopia intravital do Baço: Análise Quantitativa da Mobilidade Parasite e do fluxo sanguíneo

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Isolamento e Análise de Cérebro-sequestrados de Leucócitos<em&gt; Plasmodium berghei</em&gt; ANKA infectadas Mice

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

High Yield purificação<em&gt; Plasmodium falciparum</em&gt; Merozoítos Para uso em opsonizantes Anticorpo Ensaios

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Um<em&gt; In Vitro</em&gt; Modelo para medição de respostas imunes a Malária no Contexto do HIV co-infecção

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Análise fenotípica de roedores malária parasita assexuada e estágios do sangue sexual e estágios do mosquito

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

In Vivo Tracking of Edema Development and Microvascular Pathology em um Modelo de Malária Cerebral Experimental Usando Ressonância Magnética

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Medicina

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Medindo anticorpos indutores de fagocitose naturalmente adquiridos para parasitas plasmodium falciparum por um ensaio baseado em citometria de fluxo

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

In vitro Ensaio de Morte de Hemácias Infectadas por Plasmodium por Linfócitos Citotóxicos

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

A avaliação in vivo de Rodent Plasmodium parasitemia e Merozoito Invasion por citometria de fluxo

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