A mononuclear fraction of white blood cells (WBC) was isolated from the whole heparinized blood from 6 healthy adult pigs using a density gradient technique (Histopaque 1.077, Sigma-Aldrich, St. Louis, MO). Subsequently, a CD14-positive cell subset was selected by indirect magnetic labeling on QuadroMACS™ cell separator (Miltenyi Biotec, Gladbach, Germany) using monoclonal antibody against CD14 (clone MIL2, AbD Serotec, Oxford, UK, 10 μl per 108 cells). CD14-positive cells were captured by goat anti-mouse IgG MicroBeads (Miltenyi Biotec, Gladbach, Germany). The cell subset purity was assessed using flow cytometer LSRFortessaTM (BD Biosciences, San Jose, CA) and was more than 95% in all cases. CD14-positive monocytes, approximately 0.5 × 106 cells per well in 24-well plates, were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Paisley, UK) supplemented with antibiotics (100,000 IU/l penicillin; 10 mg/l streptomycin; 4 mg/l gentamicin) and 10% (v/v) heat-inactivated porcine serum (PAA Laboratories, Pashing, Austria) at 37°C in an atmosphere with 5% (v/v) CO2. After 6 days of cultivation, monocyte-derived macrophages (MDMF) were prepared [30 (link)].
Quadromacs cell separator
Manufactured by Miltenyi Biotec
Sourced in United Kingdom, Germany
The QuadroMACS™ cell separator is a laboratory instrument designed for the isolation and separation of specific cell populations from complex biological samples. It utilizes magnetic beads coated with antibodies that bind to target cells, allowing for efficient cell separation and purification. The QuadroMACS™ provides a standardized and automated approach to cell separation, ensuring consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Histopaque 1077, by Merck Group (3 mentions)
Goat anti mouse igg microbeads, by Miltenyi Biotec (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Ls separation column, by Miltenyi Biotec (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
Macs buffer, by Miltenyi Biotec (1 mentions)
Cd11b microbead, by Miltenyi Biotec (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
4 protocols using quadromacs cell separator
1
Isolation and Culture of Porcine Monocyte-Derived Macrophages
2
Isolation and Differentiation of Porcine Monocyte-Derived Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD14+ porcine monocytes were isolated from whole blood as described previously [31 (link)]. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Histopaque-1077 (Sigma-Aldrich) gradient. Monocytes were further enriched to a purity of >95% by positive magnetic bead selection (QuadroMACS™ cell separator, Miltenyi Biotec) using monoclonal antibody directed against CD14 (clone MIL2, AbD Serotec, Oxford, UK, 10 μL per 108 cells) and goat anti-mouse IgG microbeads together with LS separation columns (Miltenyi Biotec). The obtained cells were cultured in 24-well plates at a concentration of 5 × 105 cells per well in 1 mL of complete medium (DMEM with 10% FBS and 1% antibiotics) and incubated for 4 days at 37 °C in 5% CO2 to differentiate into macrophages. The cells for chemiluminescence assay were cultured in Nunc-Immuno™ MicroWell™ 96-well polystyrene plates (Sigma-Aldrich) at a concentration of 1 × 105 in 250 µL of complete medium and incubated for 4 days at 37 °C in 5% CO2 to differentiate into macrophages.
3
Isolation and Differentiation of Porcine Monocyte-Derived Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD14+ porcine monocytes were isolated from whole blood of six 5–6 week–old pigs as described previously [30 (link)]. Peripheral blood mononuclear cells (PBMC) were isolated from the collected whole blood by density gradient centrifugation using Histopaque−1077 (Sigma). CD14+ monocytes were purified from PBMC by staining with mouse–anti–swine CD14 (clone MIL2, AbD Serotec, Oxford, UK, 10 μl per 108 cells) and goat-anti-mouse IgG MicroBeads followed by an immunomagnetic separation method (QuadroMACS™ cell separator, Miltenyi Biotec, Gladbach, Germany). The cell subset purity was assessed using flow cytometer LSRFortessaTM (BD Biosciences, San Jose, CA) and was more than 95 % in all cases. Purified CD14+ monocytes were cultured in DMEM medium supplemented with 10 % fetal bovine serum and 1 % antibiotics (Antibiotic Antimycotic Solution 100×: 10,000 units penicillin, 10 mg streptomycin, and 25 μg amphotericin B per mL; Sigma-Aldrich) for 4 days to differentiate into macrophages (MDMF).
4
Microglia Isolation from Mixed Glia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We gently scraped and applied the cells to an antigen–antibody-mediated magnetic cell-sorting (MACS, Miltenyi Biotec) assay to positively select microglia. Briefly, the mixed glial population was re-suspended in MACS buffer (Miltenyi Biotec) and incubated with CD11b MicroBeads (Miltenyi Biotec). The cell suspension was then applied to LS separation column (Miltenyi Biotec) fitted into a QuadroMACS cell separator (Miltenyi Biotec). Unlabeled cells were allowed to pass through the column while labeled cells remained captured in the magnetic field. After washing the column with MACS buffer, the column was then removed from the magnetic separator and flushed with MACS buffer to collect the purified microglia population. For an increased level of purity, the eluted microglia population was passed through a new LS separation column a second time. The purity of microglia used in our study was more than 95% assessed by immunocytochemistry (data not shown). Microglia either acutely collected from the LS separation column or incubated on a poly-l -lysine-coated plate for 24 h were homogenized, and total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN).
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!