Mice were sacrificed by spinal dislocation and injected with 5 ml of PBS. Peritoneal macrophages acquired from ascites extraction were then isolated and purified by the MagniSort™ Mouse F4/80 Positive Selection Kit (Invitrogen). For macrophages depletion, the mice received an i.p. injection of 300 µl of clodronate liposomes (LIPOSOMA), 1 day before the experiment. Empty liposomes were used in the no-depletion group. Samples were collected 3–7 days after DSS treatment.
Magnisort mouse f4 80 positive selection kit
Manufactured by Thermo Fisher Scientific
The MagniSort Mouse F4/80 Positive Selection Kit is a laboratory equipment product designed for the isolation and purification of mouse F4/80-positive cells from a single-cell suspension. The kit utilizes magnetic beads coated with anti-F4/80 antibodies to selectively bind and separate the target cells from the sample.
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Lab products found in correlation
Dynabeads mouse t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Celltrace cfse, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse baff, by R&D Systems (1 mentions)
April, by Thermo Fisher Scientific (1 mentions)
April, by R&D Systems (1 mentions)
3t3 l1 preadipocyte, by American Type Culture Collection (1 mentions)
7 protocols using magnisort mouse f4 80 positive selection kit
1
Macrophage Depletion in DSS Colitis
2
Isolation of Heart Macrophages
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Heart macrophages were isolated using the MagniSort™ Mouse F4/80 Positive Selection Kit (Invitrogen, Cat. # 8802-6863) according to the manual. Briefly, a single-cell suspension of the heart was prepared as described above. Then the cells were incubated with biotinylated F4/80 selection antibodies for 10 minutes at RT, followed by centrifugation at 300g for 5 minutes in a 12 X 75 mm, 5 ml tube. The cell pellets were resuspended in cell separation buffer and incubated with magnetic beads for 10 minutes at RT. The tube containing samples was placed in the magnet for 5 minutes, and the supernatant was discarded. After total three times of positive selections in the magnet and washing with cell separation buffer, cells were collected by centrifugation and ready for further experiments.
3
T Cell Proliferation Assay with TAMs
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Following tumor digestion, mouse TAMs (CD11b+ F4/80+ ) were isolated and purified using the MagniSort Mouse F4/80 Positive Selection kit (Thermo Fisher Scientific). Purified T cells, isolated as described above, were stained with CellTrace CFSE (Thermo Fisher Scientific) following the manufacturer’s instruction. The labeled T cells were then plated in a 96-well plate at a density of 0.5 × 105 cells and stimulated with Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher Scientific) in the presence of IL-2 (50 U/mL, PeproTech). TAMs were added at the indicated ratios. After incubation for 72–96 hours, T cells were harvested, stained with an anti-CD8 antibody, and analyzed by flow cytometry. The percentage and number of proliferating cells was calculated using FlowJo.
4
Isolation of F4/80+ Macrophages from Pancreas
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Pancreas was minced into small fragments and incubated in collagenase solution (collagenase type I [45 U/ml], collagenase type II [15 U/ml], collagenase type III [45 U/ml], collagenase type IV [45 U/ml], elastase [0.08 U/ml], hyaluronidase [30 U/ml], and DNAse type I [25 U/ml] in RPMI-1640) on a shaker at 37 °C for 40 min. Dissociated cells were passed through a 70-μm cell strainer and washed twice in phosphate-buffered saline media. A MagniSort Mouse F4/80 Positive Selection Kit (#8802-6863-74, Thermo Fisher Scientific) was then used for the magnetic separation of F4/80+ cells in single-cell suspensions by positive selection.
5
Isolation and Culture of Macrophages and Adipocytes
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For isolation of peritoneal Mϕs, peritoneal exudate cells were harvested and F4/80+ cells were purified using the MagniSort Mouse F4/80 Positive Selection Kit (eBioscience). For VAT Mϕ isolation, SVCs were firstly separated with 30 and 70% Percoll gradient and VAT Mϕs were isolated using the F4/80 positive selection kit. Conditioned media (CM) were collected from Mϕs cultured in DMEM/F12 medium for 2 days. 3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA) and freshly isolated SAT mesenchymal cells were cultured in DMEM. The differentiation and staining of adipocytes were performed as described by Alexaki et al. (11 (link)). For BAFF and APRIL stimulation, 100 ng/mL recombinant mouse BAFF (R&D Systems, Minneapolis, MN) or APRIL (Peprotech, Rocky Hill, NJ) was added into differentiation and maintenance medium.
6
Isolation and Enrichment of Macrophages from Murine Tissues
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SVF from lean and fat-fed mice (n=5 for the fat-fed mice and n= 8 for the control mice) were prepared as described above. SVF were treated with erythrocyte lysis buffer and washed two times in PBS. Positive selection to enrich for F4/80 cells was performed using the MagniSort Mouse F4/80 positive selection kit (eBioscience, Inc,). Briefly, 108 cells were suspended in a separation buffer and incubated with the selection antibody for 60 min. Cells were washed and selection beads were added to the suspension. Following sorting, cells were washed and protein homogenates were immediately prepared.
7
Isolation of Lung Alveolar Macrophages
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First, mouse lung mononuclear cells (MNCs) at a concentration of 1 × 10 8 cells/mL were introduced into a single-cell suspension. CD11c + F4/80 + lung AMs were separated from lung MNCs based on the protocol provided by the MagniSort mouse CD11c positive selection kit and MagniSort mouse F4/80 positive selection kit, which were all purchased from eBioscience Inc (an Affymetrix company, part of Thermo Fisher Scientific, Vienna, Austria). Flow cytometry analysis revealed that the purity of the cell preparation was greater than 95%.
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