PMN depletion was induced using RB6-8C5 monoclonal antibody (Ly-6G/Gr-1 specific) (eBioscience, San Diego, CA) (33 (link)). At approximately 16 h before performing shock or sham operation, 10 μg of the anti-mouse Ly-6G/Gr1 antibody or control antibody (rabbit anti-mouse IgG; Sigma-Aldrich) was administered i.p. to mice in 100 μl saline. Our previous studies have shown that during the period of 16 to 24 h after injection of anti-mouse Ly-6G/Gr1 antibody the circulating PMN count in the antibody-treated group was decreased to 0.08 ± 0.02% of total white blood cells vs. 22.2 ± 1.9% in the control group (28 (link)). There were no statistically significant differences in the number of peripheral lymphocytes, atypical lymphocytes, monocytes, or eosinophils between the antibody-treated and control groups (28 (link)). In order to determine the role of PMN NAD(P)H oxidase in interacting AMϕ, PMN repletion in neutropenic mice was performed by tail vein injection of PMN (∼2 × 106 cells) isolated from the blood of WT or gp91phox-/- mice that were subjected to either HS or sham operation. An immunomagnetic separation system (BD Biosciences Pharmingen, San Diego, CA) (34 (link)) was used to isolate PMN. Viability of the isolated PMN was > 95%, and PMN purity was >95% as assessed by trypan blue exclusion and Wright-Giemsa staining, respectively.
Immunomagnetic separation system
Manufactured by BD
Sourced in United States
The Immunomagnetic Separation System is a laboratory equipment designed for the isolation and purification of specific cell types, proteins, or other target molecules from complex biological samples. The system utilizes magnetic beads coated with antibodies or other affinity ligands to selectively bind and capture the desired target, which can then be separated from the rest of the sample using a magnetic field. The core function of this system is to provide a reliable and efficient method for the isolation and enrichment of target analytes for further analysis or downstream applications.
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Lab products found in correlation
7 protocols using immunomagnetic separation system
1
Neutrophil Depletion and Repletion Technique
2
Isolation and Purification of Mouse Lung Endothelial Cells
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MLECs were isolated as described previously42 (link). Briefly, mice were anesthetized, chest cavity was opened, and blood was removed by infusing 10 ml PBS. Lung tissues were diced into ~1 mm3 pieces and cultured in a 60 mm culture dish in growth medium (DMEM containing 2 mM glutamine, 10% FBS, 5% human serum, 50 μg/ml penicillin/streptomycin, 5 μg/ml heparin, and 80 μg/ml EC growth supplement from bovine brain) at 37 °C with 5% CO2 for 60 h. Then, remove tissue dices from dish and culture the adherent cells for 3 days. Purify the MLEC by biotin-conjugated rat anti-mouse CD31 mAb and BD IMag streptavidin particles plus-DM, and the immunomagnetic separation system (BD Biosciences) following the manufacturer’s instructions. Purified MLECs were characterized by their cobblestone morphology.
3
Isolation and Purification of Murine Neutrophils
4
Neutrophil Adhesion Assay with MLVEC
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The assay was performed as previously described.52 (link) An immunomagnetic separation system (BD Biosciences Pharmingen)53 (link) was used to isolate PMNs. Viability of the isolated PMNs was >95%, and PMN purity was >95% as assessed by trypan blue exclusion and Wright-Giemsa staining, respectively. MLVEC from WT, Nlrp3−/−, and RAGE−/− mice were sequentially treated with HMGB1 (0.5 μg/ml) for 4 h and then with LPS (1 μg/ml) for 0–24 h. After treatment, the cells were incubated with PMNs derived from WT mice for 30 min. The non-adherent neutrophils were removed with gentle washing, and the percent of adherent PMN was calculated.
5
Isolation of Alveolar Macrophages from BAL
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BAL was performed as previously described22 (link). The immunomagnetic separation system (BD Biosciences Pharmingen, San Diego, CA) was used to isolate AM from BAL fluid. Magnetic nanoparticle-conjugated antibodies (anti-mouse Gr-1, anti-CD4, anti-CD8, and anti-CD45R/B220 antibodies; BD Biosciences Pharmingen, San Diego, CA) were chosen to label and remove PMN and lymphocytes. The resulting cells consisted of >98% macrophages, and cell viability was >95%.
6
Isolation of Alveolar Macrophages from BAL
7
Isolation of Murine Polymorphonuclear Cells
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