P. falciparum 3D7 erythrocytic asexual cultures were maintained at 5% hematocrit in complete media (RPMI 1640, 25 mM HEPES, 10 μg/ml gentamycin, 0.5 mM hypoxanthine, pH 6.75), supplemented with 25 mM sodium bicarbonate and 0.5% Albumax II at 5% oxygen, 5% carbon dioxide and 90% nitrogen. Parasite cultures were synchronized using magnetic separation of schizont stages with MACS cell separation column (Miltenyi Biotec). P. falciparum infected erythrocytes in late stages were added to 96-well plates at 1.9% parasitemia 5% hematocrite and incubated for 24 h in the presence of different concentrations of Ang II (Bachem Americas, Inc., CA, USA). 10 μl from each well were smeared on glass slides and stained with Giemsa before blind microscopic quantification of parasites. To asses whether Ang II interferes in the process of erythrocyte rupture after the completion of the infection cycle, 5x105 schizont stages per well (96% purity) were added to 96-well plates in the presence of different concentrations of Ang II (n = 8/dose) and incubated for 16 h and therefore analyzed independently to quantify the remaining non-ruptured erythrocytes in a standard hemocytometer.
Macs cell separation column
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
MACS cell separation columns are used for the isolation and purification of specific cell types from complex biological samples. They utilize paramagnetic microbeads coated with antibodies or ligands that bind to target cells, allowing for their separation in a magnetic field. The columns are designed to provide efficient and reliable cell separation, enabling the enrichment of desired cell populations for various research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Lithium heparin tube, by BD (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Lymphoprep, by Axis-Shield (2 mentions)
Mycoalert mycoplasma detection kit, by Lonza (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Angii, by Bachem (1 mentions)
Fibronectin, by R&D Systems (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Cd3 cd28 dynabead, by Thermo Fisher Scientific (1 mentions)
Cd3 microbeads, by Miltenyi Biotec (1 mentions)
Aid elispot reader classic elr 07, by Autoimmun Diagnostika (1 mentions)
Bleomycin, by Merck Group (1 mentions)
Cytofix cytoperm and perm wash, by BD (1 mentions)
Anti mouse cd45 magnetic beads, by Miltenyi Biotec (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Collagenase a, by Roche (1 mentions)
22 protocols using macs cell separation column
1
Investigating Angiotensin II's Impact on Plasmodium falciparum Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
P. falciparum 3D7 erythrocytic asexual cultures were maintained at 5% hematocrit in complete media (RPMI 1640, 25 mM HEPES, 10 μg/ml gentamycin, 0.5 mM hypoxanthine, pH 6.75), supplemented with 25 mM sodium bicarbonate and 0.5% Albumax II at 5% oxygen, 5% carbon dioxide and 90% nitrogen. Parasite cultures were synchronized using magnetic separation of schizont stages with MACS cell separation column (Miltenyi Biotec). P. falciparum infected erythrocytes in late stages were added to 96-well plates at 1.9% parasitemia 5% hematocrite and incubated for 24 h in the presence of different concentrations of Ang II (Bachem Americas, Inc., CA, USA). 10 μl from each well were smeared on glass slides and stained with Giemsa before blind microscopic quantification of parasites. To asses whether Ang II interferes in the process of erythrocyte rupture after the completion of the infection cycle, 5x105 schizont stages per well (96% purity) were added to 96-well plates in the presence of different concentrations of Ang II (n = 8/dose) and incubated for 16 h and therefore analyzed independently to quantify the remaining non-ruptured erythrocytes in a standard hemocytometer.
+ Expand
P. falciparum 3D7 erythrocytic asexual cultures were maintained at 5% hematocrit in complete media (RPMI 1640, 25 mM HEPES, 10 μg/ml gentamycin, 0.5 mM hypoxanthine, pH 6.75), supplemented with 25 mM sodium bicarbonate and 0.5% Albumax II at 5% oxygen, 5% carbon dioxide and 90% nitrogen. Parasite cultures were synchronized using magnetic separation of schizont stages with MACS cell separation column (Miltenyi Biotec). P. falciparum infected erythrocytes in late stages were added to 96-well plates at 1.9% parasitemia 5% hematocrite and incubated for 24 h in the presence of different concentrations of Ang II (Bachem Americas, Inc., CA, USA). 10 μl from each well were smeared on glass slides and stained with Giemsa before blind microscopic quantification of parasites. To asses whether Ang II interferes in the process of erythrocyte rupture after the completion of the infection cycle, 5x105 schizont stages per well (96% purity) were added to 96-well plates in the presence of different concentrations of Ang II (n = 8/dose) and incubated for 16 h and therefore analyzed independently to quantify the remaining non-ruptured erythrocytes in a standard hemocytometer.
2
In Vitro Cultivation of P. falciparum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The erythrocytic asexual stage of P. falciparum (3D7 strain) was cultured in 96 well plates for 96 h, in RPMI media containing 25 mM HEPES, 25 mM sodium bicarbonate, 10 μg/ml gentamycin, 0.5 mM hypoxanthine, at pH 6.75 in atmospheric conditions (5% CO2, 5% O2, and 90% N2). Alternative cultures were done in the presence of DON (50 μM). To set up the culture the parasites were synchronized using MACS cell separation column (MiltenyiBiotec) and the parasitemia was maintained below 5%. The parasite load was determined by GIEMSA staining of peripheral blood smear.
3
T Cell Chemotaxis Assay with CFSE
4
Antigen-Specific T Cell Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD8+ T cells derived from cancer patients with known NY-ESO-1/MAGE-A3 tumor antigen expression were purified with a MACS cell separation column (Miltenyi Biotec, Lund, Sweden) and presensitized with peptide-pulsed (10 μg/mL), irradiated, autologous peripheral blood mononuclear cells (PBMCs) depleted of CD4+ and CD8+ T cells. Presensitized CD8+ T cells were tested on days 10–12 by the IFN-γ ELISpot assay for recognition of peptide-pulsed (1 μg/mL), autologous APC dendritic cells (DCs; or Epstein-Barr virus [EBV]-transformed B cells). The number of cytokine-producing, antigen-specific T cells was evaluated using the AID ELISpot Reader Classic ELR 07 (Autoimmun Diagnostika, Strassberg, Germany). The number of presensitized effector cells was 2.5 × 104 cells per well (0.5 × 104 for a T cell clone), and ELISpot incubation time was 16 h.
5
Isolation and Analysis of Immune Cells
6
PBMC Isolation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMC experiments were carried out with ethical approval. Demographics of healthy donors are shown in Table 1 . Peripheral blood samples (approx. 50 ml) were drawn into lithium heparin tubes (Becton Dickinson). Samples were maintained at room temperature and processed immediately the following venepuncture. PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque. Proliferation assays were performed immediately after PBMC isolation. The median number of PBMCs routinely derived from healthy donors was 1.36 × 106 PBMC/ml whole blood (range 0.6 × 106–1.8 x 106/ml). The median viability as assessed by trypan blue exclusion was 90.6% (range 80–97%). For CD25 depletion PBMCs were processed as above and then immediately enriched with anti-CD25 microbeads and MACS cell separation columns (Miltenyi).
7
Monocyte-Derived Macrophage Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells were isolated from a buffycoat (Sanquin blood supply, Amsterdam, the Netherlands) through density centrifugation using Lymphoprep™ (Axis‐Shield, Dundee, Scotland). Monocytes were then purified using human CD14 magnetic beads and MACS® cell separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes were plated in 24‐well tissue culture plates at a density of 1×106 cells/mL (500 μL per well) and differentiated to macrophages for 6 days in Iscove's Modified Dulbecco's Medium (Sigma‐Aldrich) supplemented with 2 mmol/L l ‐glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL) and 10% fetal calf serum (All Gibco, Waltham, MA) in the presence of 50 ng/mL macrophage colony stimulating factor (MCSF) (Miltenyi Biotec, Bergisch Gladbach, Germany). On day 3, the medium was removed and substituted by fresh Iscove's Modified Dulbecco's Medium with 10% fetal calf serum and 50 ng/mL MCSF. On day 6, all medium was removed and replaced by Iscove's Modified Dulbecco's Medium with 10% fetal calf serum without MCSF and cells were activated for 18 hours with vehicle (DMSO), LPS (10 ng/mL, Sigma‐Aldrich), a liver X receptor (LXR) agonist (TO‐901317, 10 μmol/L, Sigma‐Aldrich), and LPS+LXR agonist. Total RNA was extracted from the cell lysate.
8
Formulation and Characterization of SPIO Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified Miglyol 840 (oil phase) was kindly provided by IOI OLEO GmbH (Hamburg, Germany). Egg lecithin containing 82.3% phosphatidylcholine (Lipoid E80) and N-(carbonyl-methoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE–PEG2000) were provided by Lipoid GmbH (Ludwigshafen, Germany); polysorbate 80 (Tween 80) was purchased from SEPPIC (Paris, France). The hetero-bifunctional linker 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-maleimide (DSPE–PEG3400–maleimide) was purchased from Laysan Bio, Inc. (Arab, AL 35016, USA). SPIO nanoparticles were synthesized and made hydrophobic by following previously described procedures [65 (link),66 (link),67 (link)]. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; ≥98%), hydrochloric acid, formic acid, the PBS buffer, sodium hydroxide, and IgG were bought from Sigma-Aldrich (St. Louis, MO 63178, USA). The PBS–heparin solution was from Sanofi Aventis (Vitry-sur-Seine, France). Glycerol was purchased from Cooperation Pharmaceutique Française (Melun, France). MACS Cell Separation Columns were from Miltenyi Biotec (Bergisch Gladbach, Germany).
9
Isolation and Analysis of Endothelial and Smooth Muscle Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from purified ECs isolated from lungs. Lungs of the mice were extracted and then incubated for 1 hour with collagenase IV 2 mg/mL at 37°. After centrifugation at 4200 rpm for 7 minutes, cells were labeled using rat anti-CD31 antibodies (BD Pharmingen Inc) then purified using anti-rat IgG MicroBeads (Miltenyi Biotec) and MACS Cell Separation Columns (Miltenyi Biotec) according to the manufacturer's instruction. SMCs were isolated from the aorta: endothelium was mechanically removed by swabbing the lumen with a cotton swab then the adventicia was mechanically torn apart after a 10-minute treatment with collagenase-1 (1 mg/mL). Mouse tissues or cells were homogenized in TRI-REAGENT (Thermofisher) and RNA extracted (Sewduth et al, 2014). For quantitative reverse-transcription polymerase chain reaction analyses, total RNA was reverse transcribed with Moloney Murine leukemia virus reverse transcriptase (Promega), and amplification was performed on a DNA Engine Opticon2 (MJ Research Inc) using B-R SYBER Green SuperMix (Quanta Biosciences). Primer location on Phactr1 sequence is shown in Figure 1 and sequences are reported in Table S1. The relative expression of each mRNA was calculated by the comparative threshold cycle method and normalized to β-actin mRNA expression. 19 (link)
10
Isolation and Characterization of Murine Immune Cell Subsets
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!