Tumor tissues were collected and cut into small pieces in PBS. After centrifugation, enzyme digestion was performed with 1 mg ml−1 collagenase (Sigma-Aldrich), 2 units ml−1 hyaluronidase (Sigma-Aldrich), and 0.1 mg ml−1 DNase (Sigma-Aldrich) for 1 h. Then the cell suspension was centrifuged with Ficoll to get the mononuclear cells and/or sorted with anti-F4/80 microbeads (Miltenyi Biotec) to get tumor-infiltrating macrophages. For tumor-infiltrating lymphocyte and macrophage isolation in tumor ascites, ascites volume and the CD45− cells (H22 tumor cell number) were counted, ascites were centrifuged with Ficoll directly to get mononuclear cells, and then sorted with anti-F4/80 microbeads (Miltenyi Biotec) to get tumor-infiltrating macrophages.
Anti f4 80 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
Anti-F4/80 microbeads are a laboratory tool used for the isolation and enrichment of F4/80-positive cells from various biological samples. They consist of magnetic beads coated with antibodies specific to the F4/80 antigen, which is a marker for mature macrophages. These microbeads can be used in cell separation and purification protocols to facilitate the study of macrophage biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Hyaluronidase, by Merck Group (4 mentions)
Macs separator, by Miltenyi Biotec (4 mentions)
Anti cd146 microbeads, by Miltenyi Biotec (3 mentions)
Collagenase, by Merck Group (3 mentions)
Dnase, by Merck Group (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (2 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Percoll, by GE Healthcare (2 mentions)
Anti cd11c microbeads, by Miltenyi Biotec (1 mentions)
Recombinant murine gm csf, by Thermo Fisher Scientific (1 mentions)
Magnetic separation column, by Miltenyi Biotec (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Mouse cd8a ly 2 microbeads, by Miltenyi Biotec (1 mentions)
Cm m040, by Procell (1 mentions)
Dnase 1, by Roche (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Collagenase a, by Merck Group (1 mentions)
Percoll, by Merck Group (1 mentions)
34 protocols using anti f4 80 microbeads
1
Tumor-Infiltrating Immune Cell Isolation
2
Tumor-Infiltrating Immune Cell Isolation
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Tumour tissues were collected and cut into small pieces in PBS, enzyme digestion was performed with 2 units mL−1 hyaluronidase (Sigma-Aldrich), 1 mg mL−1 collagenase (Sigma-Aldrich) and 0.1 mg mL−1 DNase (Sigma-Aldrich) for 1 h after centrifugation. The cell suspension was centrifuged with Ficoll to get the mononuclear cells and/or sorted with anti-F4/80 microbeads (Miltenyi Biotec) to get tumor-infiltrating macrophages. For tumor-infiltrating lymphocyte and macrophage isolation in tumor ascites, ascites volume and the CD45− cells were counted, ascites were centrifuged with Ficoll directly to get mononuclear cells. Then sorted with anti-F4/80 microbeads (Miltenyi Biotec) to get tumor-infiltrating macrophages.
3
Murine Bone Marrow-Derived Dendritic Cell Culture
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BMDC culture was performed as previously described26 (link),36 (link). Briefly, bone marrow was flushed from the femurs and tibiae of mice using sterile HBSS. Bone marrow cells were washed, counted, and plated in complete medium (RPMI-1640 supplemented with 10% FBS, 2 mM l -glutamine, 100 U penicillin/ml, 100 μg streptomycin/ml, and 50 mM 2-mercaptoethanol) supplemented with 20 ng/ml recombinant murine GM-CSF (Peprotech, Rocky Hill, NJ) at a concentration of 2 × 105 cells/ml in 10 ml (2 × 106 cells/plate). Cells were incubated at 37 °C, 5% CO2 for a total of eight days. At day 3 of incubation, 10 ml complete medium + GM-CSF was added to each plate. At day 6 of incubation, half of the medium was removed, and 10 ml fresh complete medium + GM-CSF was added. BMDCs were harvested at day 8, and DCs were purified by negative selection using anti-F4/80 microbeads (Miltenyi Biotec, Auburn, CA) (to remove contaminating macrophages) followed by positive selection using magnetically labeled anti-CD11c microbeads according to the manufacturer’s protocol (Miltenyi Biotec). Dendritic cell purity following this procedure was > 90% by flow cytometry.
4
Macrophage Adoptive Transfer in Mice
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Donor mice were sacrificed and the spleen was harvested. Spleen immune cell was isolated and macrophages were further isolated by using magnetic bead separation methods. In short, the cell number in the single cell suspension was determined and then centrifuged. Next, the cell pellet was incubated with anti-F4/80 microbeads (130-110-443, Miltenyi Biotec) according to the manufacturer’s instructions. Recipient mice were injected intravenously with 2 × 106 macrophages. Then mice were treated with oxaliplatin after three days of injection.
5
Isolation and Purification of Hepatocytes and Liver Cells
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Hepatocytes were isolated by a two-step collagenase perfusion technique as previously described26 (link) with modifications. In brief, the mice were surgically opened under anesthesia, and the livers were perfused through the IVC with 0.5 mM EGTA, followed by perfusion with 100 ml of collagenase type IV (Wellington) in Hank’s balanced salt solution (HBSS, containing calcium and magnesium; Gibco). After the liver was digested, it was resected, cut into small pieces and passed through a 70-μm cell strainer. Hepatocytes were separated from nonparenchymal cells (NPCs) by low-speed centrifugation. The NPC fraction was further separated into Kupffer cells (labeled with anti-F4/80 microbeads, 130-110-443, Miltenyi Biotec) and liver sinus epithelial cells (labeled with anti-CD146 microbeads, 130-093-596, Miltenyi Biotec) via a magnetic separation column (Miltenyi Biotech, Auburn, CA) according to the manufacturer’s protocols.
6
Isolation and Culture of Liver Cell Populations
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Splenocyte suspensions were prepared by homogenization as described previously. 31 CD8 + T cells were purified by MACS separation using mouse CD8a (Ly-2) MicroBeads (Miltenyi Biotec). Peritoneal macrophages were collected from na€ ıve animals by RPMI-1640 lavage as described previously. 34 Bone marrow cells from tibias and femurs of mice were differentiated into dendritic cells (DCs) with GM-CSF and IL-4 as described previously. 35 Isolation of PMHs, LSECs, KCs and intrahepatic lymphocytes (IHLs) was performed by liver perfusion through the portal vein with 70 lg/ml Liberase Blendzymes in Grey's balanced saline solution at 37°C. 36 Parenchymal and non-parenchymal liver cells were separated by centrifuging at 50 g for 5 min. PMHs were washed twice with complete DMEM by centrifuging at 50 g and then plated at a density of 1 9 10 5 cells per well on collagen I-coated 24-well plate in Williams' medium supplemented with 10% fetal bovine serum (Gibco). LSECs were isolated by MACS separation using anti-CD146 MicroBeads (Miltenyi Biotec), and then, 5 9 10 5 cells per well were plated on collagen I-coated 48-well plate in DMEM supplemented with 10% fetal bovine serum. KCs were isolated by MACS separation using anti-F4/80 MicroBeads (Miltenyi Biotec), and then, 2 9 10 5 cells per well were plated on collagen I-coated 96-well plate in DMEM supplemented with 10% fetal bovine serum.
7
Isolation and Culture of Primary Liver Cells
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Primary liver cells were isolated by in situ perfusion of the liver as described previously.[11 (link)
] Then the cell suspension was filtered through a 70 µm cell strainer, and non‐hepatocytes were gathered by using density gradient centrifugation. Primary Kupffer cells were further isolated using Anti‐F4/80‐MicroBeads (130‐110‐443, Miltenyi Biotec). IF staining of F4/80 was used to identify Kupffer cells and the Kupffer cells were cultured in high‐glucose dulbecco's modified eagle medium (DMEM) solution containing 10% fetal bovine serum (FBS) with 100 U mL−1 penicillin G and 100 U mL−1 streptomycin sulfate at 37 °C with 5% CO2. Primary LSECs were isolated using Anti‐CD146‐MicroBeads (130‐092‐007, Miltenyi Biotec). IF staining of CD31 was used to identify LSECs, and LSECs were cultured in mouse LSEC complete medium (CM‐M040, Procell Life Science & Technology Co., Ltd.) at 37 °C with 5% CO2 on collagen I‐coated plates.
] Then the cell suspension was filtered through a 70 µm cell strainer, and non‐hepatocytes were gathered by using density gradient centrifugation. Primary Kupffer cells were further isolated using Anti‐F4/80‐MicroBeads (130‐110‐443, Miltenyi Biotec). IF staining of F4/80 was used to identify Kupffer cells and the Kupffer cells were cultured in high‐glucose dulbecco's modified eagle medium (DMEM) solution containing 10% fetal bovine serum (FBS) with 100 U mL−1 penicillin G and 100 U mL−1 streptomycin sulfate at 37 °C with 5% CO2. Primary LSECs were isolated using Anti‐CD146‐MicroBeads (130‐092‐007, Miltenyi Biotec). IF staining of CD31 was used to identify LSECs, and LSECs were cultured in mouse LSEC complete medium (CM‐M040, Procell Life Science & Technology Co., Ltd.) at 37 °C with 5% CO2 on collagen I‐coated plates.
8
Isolation of Lung Macrophages
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At 3 days postinfection, lungs were homogenized with a gentleMACS dissociator (Miltenyi Biotec) in 5 ml complete RPMI with 2 mg/ml collagenase D and 40 U DNase I (Roche) for 30 min at 37°C. Erythrocytes were removed using ammonium-chloride-potassium (ACK) lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA). Cells were filtered through a 40-mm nylon mesh, washed, and counted. Anti-F4/80 MicroBeads were used for positive selection of F4/80+ cells (Miltenyi Biotec).
9
Isolation of F4/80+ Murine Splenocytes
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RPMs were isolated from 1 × 108 splenocytes by positive selection with anti-F4/80 microbeads, MACS columns, and MACS separators according to the manufacturer’s protocols (Miltenyi Biotech, Bergisch Gladbach, Germany).
10
Isolating Tumor-Associated Macrophages
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For TAM isolation, xenograft tumors were collected and digested into single-cell suspensions as previously described [20] , and macrophages were isolated using Anti-F4/80 MicroBeads (Miltenyi Biotec, Germany) according to the manufacturer's instructions.
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