VarioMACS (Miltenyi Biotec France) purified IEs at mid and late trophozoites stages were resuspended in PBS 0.2% BSA and counted. For each assay, 0.5 × 106 IEs were washed in PBS and incubated with 50 μl of purified rabbit polyclonal anti-VAR2CSA IgG diluted 1/100 in PBS 0.2% BSA for 1 h at RT. IEs were washed twice with PBS and resuspended in 100μl of PE conjugated goat anti-rabbit IgG diluted 1/100 in PBS for 30 min at RT. After washing twice in PBS 0.2% BSA, IEs were resuspended in paraformaldehyde 2% in PBS and kept at 4°C overnight in darkness. Cells were then washed twice with PBS and analysed by flow cytometry using a BD FACScanto II flow cytometer (Becton Dickinson France). with the Flow Jo 10.0 software. Parasite nuclei was stained with Topro3 (1/10,000 dilution). The results are expressed as the geometric mean fluorescence intensities.
Variomacs
Manufactured by Miltenyi Biotec
Sourced in Germany, France
The VarioMACS is a compact cell separation system designed for magnetic cell separation applications. It offers a flexible and versatile platform for the isolation and purification of various cell types from diverse sample sources. The VarioMACS system utilizes magnetic beads and columns to selectively bind and separate target cells, allowing for efficient cell enrichment or depletion.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (3 mentions)
Imdm culture medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (1 mentions)
Cellgro scgm serum free medium, by CellGenix (1 mentions)
Anti cd158e pe z27.3.7, by Beckman Coulter (1 mentions)
Interleukin 2 (il 2), by Nipro (1 mentions)
A 350n culture bag, by Nipro (1 mentions)
Z1 coulter particle counter, by Beckman Coulter (1 mentions)
Cs column, by Miltenyi Biotec (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Lookout mycoplasma pcr detection kit, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Leica em ice, by Leica Microsystems (1 mentions)
Em afs2, by Leica Microsystems (1 mentions)
To pro 3, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Glycerolyte 57, by Baxter (1 mentions)
Pierce protein a agarose beads, by Thermo Fisher Scientific (1 mentions)
Gfp trap agarose beads, by Proteintech (1 mentions)
23 protocols using variomacs
1
Quantification of VAR2CSA Expression
2
Expansion and Characterization of Human NK Cells
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In vitro expansion of human NK cells was conducted as previously described [18 (link)]. Briefly, peripheral blood mononucleated cells (PBMCs) were isolated from healthy donors by leukapheresis and CD3+ T cells were depleted by VarioMACS (Miltenyi Biotech). T cell-depleted PBMCs were expanded at a seeding concentration of 2 × 105 cells/ml in CellGro SCGM serum-free medium (CellGenix) with 1 % autoplasma, 1 × 106 cells/ml irradiated (2,000 rad) autologous PBMCs, 10 ng/ml anti-CD3 monoclonal antibody, OKT3 (Orthoclon), and 500 IU/ml IL-2 (Proleukin) in an A-350 N culture bag (NIPRO). On day 5 of culture, NK cells were fed with fresh medium containing 500 IU/ml IL-2 and 1 % autoplasma every two days without removal of preexisting culture medium to maintain a cellular concentration at 1 ~ 2 × 106 cells/ml for 14 days. The viability of expanded NK cells was determined by propidium iodide staining.
In vitro expanded NK cells were stained with primary antibodies and analyzed by a flow cytometry using anti-CD56-PE-Cy5 (B159), anti-CD16-PE (3G8), anti-CD3-FITC (UCHT1), anti-NKp30-PE (P30-15), anti-NKp44-PE (P44-8.1), anti-NKp46-PE (9E2/NKp46), anti-DNAM-1-PE (DX11), anti-CD14-FITC (M5E2), anti-CD19-PE (HIB19) (BD Biosciences), anti-NKG2D-PE (149810) (R&D systems), anti-CD158a-PE (EB6Bf), anti-CD158b-PE (GL183), and anti-CD158e-PE (Z27.3.7) (Beckman Coulter).
In vitro expanded NK cells were stained with primary antibodies and analyzed by a flow cytometry using anti-CD56-PE-Cy5 (B159), anti-CD16-PE (3G8), anti-CD3-FITC (UCHT1), anti-NKp30-PE (P30-15), anti-NKp44-PE (P44-8.1), anti-NKp46-PE (9E2/NKp46), anti-DNAM-1-PE (DX11), anti-CD14-FITC (M5E2), anti-CD19-PE (HIB19) (BD Biosciences), anti-NKG2D-PE (149810) (R&D systems), anti-CD158a-PE (EB6Bf), anti-CD158b-PE (GL183), and anti-CD158e-PE (Z27.3.7) (Beckman Coulter).
3
Cellular Drug Accumulation in Malaria Parasites
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The drug accumulation assay has been fully described [21] (link), [75] (link). Briefly, P. falciparum-infected erythrocytes were purified using a strong magnet (VarioMACS, Miltenyi Biotec), as described [75] (link). This yielded a purity of trophozoite-infected erythrocytes of 95–100% as determined by light microscopic examination of Giemsa-stained blood smears. The cells were resuspended in prewarmed RPMI 1640 containing 11 mM glucose, 25 mM HEPES, and 2 mM glutamine (pH 7.3 at 37°C) at an haematocrit of 25000 cells/µl. The haematocrit was determined using an automated cell counter (Z1-Coulter Particle Counter, Beckman Coulter Inc.). Cells were then incubated at 37°C in the presence of 40 nM of the respective drug and the amount of label accumulated over time was monitored. The cellular drug accumulation ratio was determined as described [75] (link). Throughout the study, trophozoite-infected erythrocytes (20–28 hrs post invasion) were examined. The accumulation ratios to quinine, quinidine and chloroquine were determined in parallel assays.
4
Cultivation and Characterization of P. falciparum Strains
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Three P. falciparum laboratory adapted parasite lines were used in this study: NF54, FCR3 and 7G8. Erythrocytes infected by a select parasite strains and selected for CSA-binding phenotype are referred as NF54-CSA, FCR3-CSA and 7G8-CSA. One control parasite (FCR3) selected for CD36-binding phenotype is referred as FCR3-CD36. Parasites were grown in O+ human erythrocytes in RPMI 1640 medium containing Hepes (25 mM) and L-glutamine (2 mM) (Gibco) supplemented with 5% Albumax, 5% human serum, 0.1 mM hypoxanthine and 20 µg/ml gentamicin46 (link). Prior to each experiment, the CSA or CD36 -binding phenotypes of the parasitized red blood cells were verified on receptors immobilized on plastic Petri dishes as previously described47 (link). Parasites were routinely genotyped by PCR using MSP1/MSP2 primers48 (link) and tested for potential mycoplasma contamination (LookOut Mycoplasma PCR Detection Kit by SIGMA). Synchronized parasite cultures (3–7% parasitemia) at mid/late trophozoite stages were purified using VarioMACS and CS columns (Miltenyi Biotec) as previously described49 (link).
5
High-Pressure Freezing and Cryo-Imaging of P. falciparum
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Red blood cells infected with P. falciparum 3D7 expressing 3xNLS-mCherry and PCNA1::GFP in multinucleated stages were purified via varioMACS (Miltenyi Biotec GmbH) magnetic purification, washed in phosphate-buffered saline (PBS) (Gibco, Thermo Fisher Scientific), and resuspended in incomplete RPMI [0.2 mM hypoxanthine, 25 mM Hepes (pH 7.3), and gentamicin (12.5 μg/ml)]. The pellet was pipetted into 0.2-mm-deep aluminum carriers (Engineering Office M. Wohlwend GmbH) and cryo-immobilized by high-pressure freezing using Leica EM ICE (Leica Microsystems).
Freeze substitution was done using a freeze substitution device (EM-AFS2, Leica Microsystems), the freeze substitution solution contained 0.3% (w/v) uranyl acetate dissolved in anhydrous acetone, and the samples were substituted at −90°C for 14 hours. The temperature was then increased at a rate of 5°C/hour to −45°C, followed by 2-hour incubation at −45°C. The samples were rinsed with ethanol for 1 hour, followed by LR Gold (London Resin Company) infiltration at −25°C in 2-hour steps with 25, 50, and 75% LR Gold in ethanol. The samples were then left in 100% LR Gold for 12 and 4 hours before the onset of polymerization. Ultraviolet (UV) polymerization was applied for 24 hours at −25°C, and the temperature was increased to 20°C at a rate of 5°C per hour. The samples were left exposed to UV at room temperature for 24 hours.
Freeze substitution was done using a freeze substitution device (EM-AFS2, Leica Microsystems), the freeze substitution solution contained 0.3% (w/v) uranyl acetate dissolved in anhydrous acetone, and the samples were substituted at −90°C for 14 hours. The temperature was then increased at a rate of 5°C/hour to −45°C, followed by 2-hour incubation at −45°C. The samples were rinsed with ethanol for 1 hour, followed by LR Gold (London Resin Company) infiltration at −25°C in 2-hour steps with 25, 50, and 75% LR Gold in ethanol. The samples were then left in 100% LR Gold for 12 and 4 hours before the onset of polymerization. Ultraviolet (UV) polymerization was applied for 24 hours at −25°C, and the temperature was increased to 20°C at a rate of 5°C per hour. The samples were left exposed to UV at room temperature for 24 hours.
6
Purification of Natural Killer Cells
7
Immunogenicity Assay of Malaria Parasites
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VarioMACS (Miltenyi Biotec) purified IEs at mid/late trophozoite stage were resuspended in PBS 0.2% BSA and counted. For each assay, 2.5 × 105 IEs were washed in PBS and incubated with the sera (previously depleted against normal erythrocytes) diluted 1/10 in PBS 0.2% BSA for 1 h at 4 °C. IEs were washed twice with PBS and resuspended in 100 µl of PE conjugated goat anti-rabbit or goat anti-rat IgG diluted 1/100 in PBS 0.2% BSA for 60 min at 4 °C. After washing twice in PBS 0.2% BSA, IEs were resuspended in paraformaldehyde 2% in PBS and kept at 4 °C overnight in darkness. Cells were then washed twice with PBS and analyzed by flow cytometry using a BD FACScanto II flow cytometer with the Flow Jo 10.0 software. Parasite nuclei were stained with Topro3 (1/4,000 dilution) (ThermoFischer Scientific). The results are expressed as the ratio between the geometric mean fluorescence intensities (MFI) of the immune sera and the respective non-immune sera.
8
Enrichment of Synchronized Malaria Parasites
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Suspensions containing synchronized parasite cultures of t-RBCs at 4–7% parasitemia were passed through a magnetic column (MACS LS column, Miltenyi Bioc). This procedure takes advantage of the electromagnetic properties of hemozoin that retains t-RBCs and allows their separation from noninfected cells [36] .
Briefly, LS columns were mounted on a high-gradient magnetic cell separator VarioMACS (Miltenyi Biotec), and washed with 5 ml RBC medium before used. A suspension of t-RBCs (parasitemia at 4–7%) was centrifuged 900 ×g for 3 min and the pellet was suspended in 2 mL of RBC medium supplemented with 2% bovine serum albumin (BSA) and 2 mM EDTA. The suspension (2×108 t-RBCs mL-1) was loaded on and passed through the LS column, and the eluate was reloaded in the same column to optimize t-RBCs retention. The column was washed with RBC medium and removed from the magnetic field. Retained t-RBCs were eluted in RBC medium supplemented with 0.5% BSA. The parasitemia of the final suspension was 94.39±0.03% (N = 15) and subsequently denoted as t94-RBCs.
Briefly, LS columns were mounted on a high-gradient magnetic cell separator VarioMACS (Miltenyi Biotec), and washed with 5 ml RBC medium before used. A suspension of t-RBCs (parasitemia at 4–7%) was centrifuged 900 ×g for 3 min and the pellet was suspended in 2 mL of RBC medium supplemented with 2% bovine serum albumin (BSA) and 2 mM EDTA. The suspension (2×108 t-RBCs mL-1) was loaded on and passed through the LS column, and the eluate was reloaded in the same column to optimize t-RBCs retention. The column was washed with RBC medium and removed from the magnetic field. Retained t-RBCs were eluted in RBC medium supplemented with 0.5% BSA. The parasitemia of the final suspension was 94.39±0.03% (N = 15) and subsequently denoted as t94-RBCs.
9
Isolation and Purification of Malaria Trophozoites
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Trophozoites at 24–28 h post-invasion were isolated by magnetic selection with the VarioMACS (Miltenyi Biotech) system. Purity was assessed by counting 200 cells via Giemsa thin smear. Preparations varied from 70 to 99% parasite purity and preparations with purity of < 90% were re-purified by repeating magnet selection. Purified preparations were counted using a haemocytometer and resuspended to the appropriate cell concentration in complete medium (CM): RPMI1640 containing GlutaMAX (Gibco) supplemented with 10% heat-inactivated FCS, 0.1 mM 2-beta-mercaptoethanol, 100 U/mL penicillin and 100 μg/mL streptomycin. Aliquots of purified trophozoites with >95% purity were also frozen in glycerolyte 57 (Baxter Healthcare Corporation) and stored at −80°C to compare efficacy of DC stimulation with same-day isolated cultures.
10
Affinity Purification of GFP-Tagged Proteins
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Parasite-infected RBCs were purified using a VarioMACS (Miltenyi Biotec) magnet or by floatation in 70% (vol/vol) Gelofusine (Braun)–PBS. The infected RBCs were washed in RPMI medium and then solubilized on ice for 30 min in 10× pellet volumes of IP buffer (1% [vol/vol] Triton X-100, 150 mM NaCl, 50 mM Tris, 8 mM EDTA) with cOmplete protease inhibitor cocktail (Roche). The Triton X-100-insoluble material was pelleted twice by centrifugation (10 min at 16,000 × g). The supernatant was precleared with Pierce protein A agarose beads (Thermo Scientific) for 1 h at 4°C on a mixing wheel. The protein A beads were pelleted, and an aliquot of the supernatant was taken as the input fraction. The remainder of the supernatant was incubated with washed GFP-Trap agarose beads (Chromotek) for 16 h at 4°C. The GFP-Trap beads were then washed 5 times in IP buffer.
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