Freshly isolated spleens were placed in ice-cold DMEM (Life Technologies, Grand Island, NY) on a 60 mm dish. Spleens were grinded and passed through a 40-μm cell strainer. The pellet was incubated with ACK lysing buffer (Beyotime Biotechnology) for 2 min at room temperature. The renal single-cell suspensions were obtained according to Chong’s protocol (14 (link)). Then, the cells were prepared for staining and flow cytometry analysis.
Ack lysing buffer
Manufactured by Beyotime
Sourced in China
ACK lysing buffer is a solution used for the lysis of red blood cells (RBCs) in biological samples. It is a hypotonic buffer that effectively disrupts the RBC membranes, allowing for the isolation of other cellular components like white blood cells or nucleated cells. The core function of the ACK lysing buffer is to facilitate the removal of RBCs from a sample, enabling further downstream processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Ham s f12, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Solarbio (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Collagenase 4, by Worthington (1 mentions)
Collagenase, by Merck Group (1 mentions)
Anti cd11b apc, by BioLegend (1 mentions)
Anti ly6c fitc, by BioLegend (1 mentions)
Anti ly6g percp cy5, by BioLegend (1 mentions)
Anti mouse cd16 32 antibody, by BioLegend (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Percoll, by Cytiva (1 mentions)
4 protocols using ack lysing buffer
1
Isolation of Murine Splenic Cells
2
Isolation and Culture of Kidney Cells
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Freshly isolated kidneys were placed in ice-cold DMEM mixed with Hams F12 (1:1 ratio; Life Technologies, Grand Island, NY) on a 60 mm dish. The kidney capsule was removed by peeling with forceps, and the kidney was sliced coronally and homogenized by mincing into 1–2 mm3 pieces. The homogenized kidney tissue pieces were resuspended and mixed in 10 ml of collagenase type IV for 30 min at 37°C to obtain single-cell suspensions. After digestion, the cell suspension was filtered through 70-μm cell strainers. The filtered cell suspensions were centrifuged at 300g for 5 min and incubated with ACK lysing buffer (Beyotime Biotechnology, China) to remove red blood cells. Then, the pellet was washed with DMEM/F12 medium with 10% FBS twice and passed through a 40-μm cell strainer. After filtering, cells were generated in DMEM/F12 medium with 10% FBS on a 60 mm dish. Then, medium was replaced with fresh DMEM/F12 medium with 10% FBS 6 h later.
3
Isolation of Colorectal Macrophages
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Fresh CRC tissue samples were cut into pieces (<1 m3) and digested in Buffer A (PBS with 0.5% endotoxin-free BSA and 2 mM EDTA) supplemented with 1 g/L collagenase IV (#LS004188, Worthington), 0.1 g/L hyaluronidase (#H1115000, Sigma) and 0.01 g/L DNase I (#D8071, Solarbio, China). Dissociated cells were collected in a 15-ml tube and centrifuged at 400 × g for 5 min. Pellets were resuspended in ACK lysing buffer (#C3702, Beyotime, Shanghai, China) for 5 min and then washed with Buffer A before filtration with a 100-μm filter. These cells were collected to further isolate macrophages (CD45+F4/80+) by fluorescence-activated cell sorting (FACS).
4
Isolation and Identification of Myeloid Cells
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Tumors were minced and incubated for 30 min at 37°C in 2 mL digestion buffer (1 mg/mL collagenase (C2674, Sigma-Aldrich) and 100 μg/mL DNase I (D4527, Sigma-Aldrich) in RPMI 1640 medium). Cell suspensions were passed through a 100 μm cell strainer. After waching with RPMI 1640, cells were resuspended in 40% Percoll (17089101, Cytiva) and centrifuged. After centrifugation, Cells were washed with staining buffer.Spleens were minced and rinsed with 5 mM EDTA in RPMI 1640. Peripheral blood were obtained from mouse eyeballs and treated with anticoagulant reagent (G0280, Solarbio). Erythrocytes were lysed with 1 mL of ACK Lysing Buffer (C3702, Beyotime) per spleen for 2 min. Splenocytes and PBMCs were washed with RPMI 1640, centrifuged, followed by rinsing with staining buffer. After centrifugation, cells were resuspended in PBS at 1 × 108 cells/mL and incubated with anti-mouse CD16/32 antibody (101302, Biolegend) and viability dye for 15 min at RT. Then, cells were stained with anti-CD11b-APC, anti-Ly-6C-FITC (128006, Biolegend) and anti-Ly-6G-PerCP/Cy5.5 (127616, Biolegend) antibody.
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