KCs were isolated using the method described by Yue et al.20 (link) Briefly, the portal vein was exposed, a surgical thread was inserted in the portal vein and the liver was perfused with HBSS solution prewarmed at 37°C. The perfusion solution was then switched to type IV collagenase for 20 m, until the liver had slightly swelled. The liver was removed and washed with phosphate-buffered saline for three times, and then the liver was finely chopped and digested with collagenase solution for 20 m by vigorous shaking at 37°C. The resultant cell suspension was teased through a cell strainer (100 µm) and the hepatocytes in the supernatant was removed by three 2 m centrifugations at 50×g. Then the nonparenchymal cells (NPCs) were layered onto a 50%/25% two-step Percoll gradient (Sigma) and centrifuged at 2,000×g for 10 m at 4°C. KCs in the middle layer were collected.
Two step percoll gradient
Manufactured by Merck Group
Sourced in United States
The Two-step Percoll gradient is a laboratory equipment used for cell separation and purification. It consists of a density gradient prepared using Percoll, a colloidal silica suspension. The gradient allows for the separation of different cell types based on their density differences, enabling the isolation and purification of specific cell populations.
Automatically generated - may contain errors
Lab products found in correlation
70 μm nylon mesh cell strainer, by BD (3 mentions)
Collagenase 4, by Merck Group (1 mentions)
70 mm nylon mesh cell strainers, by BD (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Cytiva (1 mentions)
Mir con, by Qiagen (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Collagenase buffer, by Merck Group (1 mentions)
7 protocols using two step percoll gradient
1
Isolation of Kupffer Cells via Liver Perfusion
2
Isolation and Culture of Kupffer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KCs were isolated as previously described.59 In brief, livers were perfused in situ via the portal vein with warmed (37°C) Hanks’ balanced salt solution, followed by collagenase IV (Sigma). Perfused livers were dissected and teased through 70‐mm nylon mesh cell strainers (BD Biosciences, San Diego, CA, USA). Nonparenchymal cells were separated from hepatocytes by three 2‐min centrifugations at 50 g. Nonparenchymal cells were suspended in Hanks’ balanced salt solution and layered onto a 50%/25% two‐step Percoll gradient (Sigma) and centrifuged at 1800g at 4°C for 15 min. KCs in the middle layer were collected and allowed to attach to cell culture plates in Dulbecco's Modified Eagle Medium with 10% FBS, 10 mm (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid) (HEPES), 100 U mL−1 penicillin, 100 μg mL−1 streptomycin and 2 mm glutamine at 37°C for 15 min. Nonadherent cells were removed by replacing the culture medium. KCs were cultured for 6 h in vitro. Cells and supernatants were collected for further experiments.
3
Isolation of Mouse Kupffer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kupffer cells were separated from C57BL/6 mice according to a reference by Li et al. (2021) (link). In brief, livers of mice were perfused with 10 mL calcium-free Hank balanced salt solution (HBSS; HyClone Laboratories, San Angelo, TX, USA) via the portal vein, and then with 0.27% type IV collagenase (Sigma-Aldrich, St. Louis, MO, USA) in a water bath for 20 min at 37 °C. Subsequently, perfused livers were dissected, and single-cell suspension was prepared using a 70 mm cell filter. Hepatocytes and nonparenchymal cells were separated from cell suspension by centrifuging at 50×g and 4 °C for 3 min. Nonparenchymal cell-containing solution was suspended with HBSS, gently overlaid onto a two-step Percoll gradient (Sigma-Aldrich, St. Louis, MO, USA), and then centrifuged at 1,800×g and 4 °C for 15 min without break. Cells in the middle layer (Kupffer cells) were collected and cultured into Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin/streptomycin, and 2 mM L-glutamine at 37 °C with 5% CO2. After incubation for 2 h, the isolated Kupffer cells were purified by removing the non-adherent cells.
4
Transfection of miR-339-5p and PTP4A1 in U251 Glioma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U251 cells (The Jackson Laboratory) were cultivated with 10% fetal bovine serum DMEM medium with 100 ng/mL streptomycin and 100 U/mL penicillin in advance and placed in an incubator with 5%CO2 at 37°C. The cells were layered onto a 50%/25% two-step Percoll gradient (Sigma) in a 50-ml conical centrifuge tube and centrifuged. After the cells were overgrown, the sterile PBS was cleaned, and 0.25% trypsin was added for digestion. Follow-up experiments were conducted when the cells were in a good growth state and the logarithmic phase. Then, they were divided into NC group, miR-con group, miR-339-5p group, and miR-339-5p +PTP4A1 group, and transfection reagent Lipofectamine 3000 was applied. miR-NC was transfected into U251 as NC group, miR-con was transfected into U251 as a miR-con group, and miR-339-5p was transfected. Mimic (Qiagen, Chatsworth, CA) was transfected into U251 as a miR-339-5p group, miR-con (Qiagen, Chatsworth, CA) and PCDNA3.1-PTP4A1 (Qiagen, Chatsworth, CA) were transfected into U251 as miR-con +PTP4A1 group, and miR-339-5p was transfected. Mimic and PCDNA3.1-PTP4A1 were transfected into U251 as miR-339-5p +PTP4A1 group, respectively.
5
Isolation and Culture of Primary Hepatic Cells
6
Isolation of Hepatocytes and Kupffer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary hepatocytes and liver macrophages (Kupffer cells) from Foxo1FL/FL and Foxo1M-KO mice were isolated, as described [5 (link)]. In brief, livers were perfused in situ with warmed (37 °C) HBSS solution, followed by a collagenase buffer (collagenase type IV, Sigma-Aldrich). Perfused livers were dissected and teased through 70-μm nylon mesh cell strainers (BD Biosciences, San Jose, CA). Nonparenchymal cells (NPCs) were separated from hepatocytes by centrifuging at 50 × g for 2 min three times. NPCs were suspended in HBSS and layered onto a 50%/25% two-step Percoll gradient (Sigma) in a 50-ml conical centrifuge tube and centrifuged at 1800 × g at 4 °C for 15 min. Kupffer cells in the middle layer were collected and allowed to attach onto cell culture plates in DMEM with 10% FBS, 10 mM HEPES, 2 mM GlutaMax, 100 U/ml penicillin, and 100 μg/ml streptomycin for 15 min at 37 °C. Nonadherent cells were removed by replacing the culture medium.
7
Notch1 Regulation of Macrophage HSF1 and Snail
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine primary hepatocytes and bone-derived macrophages (BMMs) were isolated from Notch1FL/FL and Notch1M-KO mice as described.2 (link) In brief, livers were perfused in situ with warmed (37˚C) HBSS solution followed by collagenase buffer (collagenase type IV, Sigma, St Louis, MO). The perfused livers were dissected and strained through 70-μm nylon mesh cell strainers (BD Biosciences). The nonparenchymal cells (NPCs) were separated from the hepatocytes by centrifugation three times at 50 × g for 2 min. The NPCs were suspended in HBSS and layered onto a 50%/25% two-step Percoll gradient (Sigma) in a 50-ml conical centrifuge tube and centrifuged at 1800 × g at 4 °C for 15 min. Bone marrow cells were removed from the femurs and tibias of the Notch1FL/FL and Notch1M-KO mice and cultured in DMEM supplemented with 10% FBS and 15% L929-conditioned medium. BMMs (1 × 106/well) were cultured for 7 days and then transfected with CRISPR/Cas9-mediated HSF1 KO (p-CRISPR-HSF1 KO), CRISPR/Cas9-mediated Snail KO (p-CRISPR-Snail KO), CRISPR/Cas9-mediated Snail activation (p-CRISPR-Snail) or control vector. After 48 h, the cells were treated with 2 µg/ml JAG1 supplemented with 100 ng/ml LPS for an additional 6 h.
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!