Parasites from the 3D7A and the DynGFP/P47mCherry lines were induced to produce gametocytes by parasite overgrowth. After a 5-day treatment with 50 mM N-acetyl-glucosamine (NAG) to eliminate residual asexual parasites, stage III/IV gametocytes were partially purified from uninfected erythrocytes on MACS Separation Columns CS (Miltenyi Biotec). Gametocytes were then put back in culture and allowed to mature to stage V over the following 7 days. Aliquots of 5 × 105 mature gametocytes were centrifuged and pellets were stored at − 80 °C for further analyses.
Macs separation columns cs
Manufactured by Miltenyi Biotec
Sourced in Italy
The MACS Separation Columns CS are high-performance magnetic cell separation columns designed for efficient and gentle isolation of target cells from complex samples. The columns utilize the MACS technology to provide a reliable and versatile solution for cell separation applications.
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6 protocols using macs separation columns cs
1
Gametocyte Production and Purification
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Gametocyte Maturation and Drug Screening
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Radiolabeling of Plasmodium Schizonts
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Synchronous cultures of P. falciparum with 10% parasitaemia of schizonts were treated with 6.5 µM and 3.75 µM, for 4a and 4c , respectively, corresponding to 80% of IC50. A flask was kept untreated and is considered the positive control. After 36 h, the culture was labeled with [1-(n)-3H]GGPP (3.125 µCi/ml) in normal RPMI 1640 medium [32 (link)] during the last 16 h. The schizonts were purified by magnetic column (MACS Separation Columns “CS”, Miltenyi Biotec) [33 (link)]. The parasitaemia was estimated by microscopic examination of Giemsa-stained smears and the volume was adjusted of purified culture, according to the control, so that equal numbers of treated and untreated parasites were applied. After metabolic labelling and purification, lyophilized schizonts were used for extraction of MQ and TC. The final extract was injected into the HPLC-RT [34 (link)].
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Purification and Separation of Malaria Gametocytes
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Parasites from the PfDynGFP/P47mCherry line were induced to produce gametocytes as above and stage III/IV gametocytes were partially purified from uninfected erythrocytes on MACS Separation Columns CS (Miltenyi Biotec). Gametocytes were resuspended (10 × 106/ml) in 1× PBS and male and female gametocytes were separated by sorting at room temperature using a BD FACSAria flow cytometer (Becton–Dickinson). First, gametocytes were separated from uninfected erythrocytes using forward and sideward scatter and then the GFP+ (male) gametocytes were separated from the GFP− (female) gametocytes. Aliquots of the sorted samples were analysed by UV fluorescence microscopy and Giemsa stained to determine the purity of the gametocyte populations. The GFP+ and GFP− gametocytes were put back in separate cultures and were allowed to mature to stage V over the following 7 days. Mature gametocytes were counted in a haemocytometer chamber and aliquots of male and female gametocytes were centrifuged and resuspended in 50 µl medium and then in 450 µl of RNAlater.
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Gametocyte Purification from P. falciparum
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The P. falciparum 3D7 (Walliker et al., 1987 (link)) line and the Pf2004/164-tdTom line (Brancucci et al., 2015 (link)) were cultured in human 0+ erythrocytes, kindly provided by Prof. A. Angeloni, Immunohematology and Transfusion Medicine, Policlinico Umberto I, Sapienza University of Rome, at 5% hematocrit under 5% CO2, 2% O2, 93% N2 (Trager and Jensen, 1976 (link)). Cultures were grown in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 25 mM Hepes, 50 µg/ml hypoxanthine, 0.25 mM NaHCO3, 50 µg/ml gentamicin sulfate and 10% pooled heat-inactivated 0+ human serum (IBB, Memphis, TN, USA).
Cultures were induced to produce gametocytes via parasite overgrowth; after appearance of the oat shaped stage I gametocytes a 72h, treatment with 50 mM N-acetyl-glucosamine (NAG) was applied to eliminate residual asexual parasites (Gupta et al., 1985 (link)). Stage II-III gametocytes were purified from uninfected erythrocytes on MACS Separation Columns CS (Miltenyi Biotec Bologna, Italy) as previously reported (Ribaut et al., 2008 (link)).
Cultures were induced to produce gametocytes via parasite overgrowth; after appearance of the oat shaped stage I gametocytes a 72h, treatment with 50 mM N-acetyl-glucosamine (NAG) was applied to eliminate residual asexual parasites (Gupta et al., 1985 (link)). Stage II-III gametocytes were purified from uninfected erythrocytes on MACS Separation Columns CS (Miltenyi Biotec Bologna, Italy) as previously reported (Ribaut et al., 2008 (link)).
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Antimalarial Drug Screening on Asexual Parasites and Gametocytes
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