Primary hepatocytes were isolated using a two-step perfusion method. Briefly, pre-perfusion with HANKS’s buffer (HBSS supplemented with 0.5 mM EDTA, 25 mM HEPES) was performed by inserting the cannula through the superior vena cava and cutting the portal vein. Next, livers were perfused at low flow for approximately 10 min with Digestion Buffer (low glucose DMEM supplemented with 1 mM HEPES) containing freshly added Liberase TM (32 μg/mL; Roche). Digestion was stopped using Isolation Buffer (low glucose DMEM supplemented with 10% FBS) and cells were separated from the matrix by gently pushing with a cell scraper. The cell suspension was filtered through a 100 μm filter (Corning) and hepatocytes were purified by two low speed centrifugation steps (50×g for 2 min).
100 μm filter
Manufactured by Corning
Sourced in United States
The 100 μm filter is a lab equipment product designed to filter particles or substances based on size. It has a pore size of 100 micrometers, allowing it to separate materials of different dimensions. The filter's core function is to provide a physical barrier for the selective removal or retention of components from a fluid or suspension.
Automatically generated - may contain errors
Lab products found in correlation
D glucose, by Merck Group (2 mentions)
Collagenase type 1 from clostridium histolyticum, by Merck Group (2 mentions)
Liberase, by Roche (1 mentions)
Negative magnetic selection, by STEMCELL (1 mentions)
70 μm filter, by Corning (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (1 mentions)
Collagenase type 4, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Anti f4 80 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd86 fitc, by Thermo Fisher Scientific (1 mentions)
Ack buffer, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline pbs, by Biosharp (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Hanks balanced salt solution (hbss), by Corning (1 mentions)
Dnase 1, by Roche (1 mentions)
Lymphocyte separation medium, by Corning (1 mentions)
9 protocols using 100 μm filter
1
Isolation of Primary Hepatocytes
2
Isolation of CD4+ T Cells from Infant and Adult Mice
3
Establishment of Ovarian Cancer PDOs
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Fresh tumor tissues obtained from EOC patients were immersed in Advanced DMEM/F12 (Cat. 12634028, Thermo Fisher Scientific) containing 1% penicillin–streptomycin (PS). The necrotic tissues were removed, and the remaining tissue was cut into pieces of approximately 1–2 mm3 volume. After washing twice with PBS containing 1% PS, the tissue fragments were digested at 37 °C for 1 h using the Advanced DMEM/F12 solution containing type IV Collagenase (Cat. 17104019, Thermo Fisher Scientific). The above solution was filtered using a 100 μm filter (Cat. 352360, Corning, New York, USA). Next, the fragments were centrifuged at 300×g for 5 min. The erythrocytes were lysed. The precipitated cells were resuspended in the Advanced DMEM/F12 mixed with growth factor-reduced Matrigel (Cat. CB-40230, Corning, USA). The cell suspension was adjusted to a final concentration of 10,000–20,000 cells/50 μl. PDOs were transferred to 24-well plates when the Matrigel solidified, and 500 μl of the general culture medium was added. PDOs were grown in a cell incubator at 37 °C with 5% CO2. The culture medium was changed every 2–3 days. The cell viability of PDOs was determined by the Cell Counting-Lite 3D Luminescent Cell Viability Assay (Cat. DD1102-01, Vazyme, Nanjing, China).
4
Macrophage Polarization Assessment
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The distribution of CD86 and CD206 was detected using flow cytometry to determine the ratio of M1/M2 macrophages. Lung single-cell suspension was obtained using a 100-μm filter (Corning Glass Works, Corning, NY, USA) treated with ACK buffer (Thermo Fisher Scientific). Anti-F4/80-APC, anti-CD86-FITC, anti-CD206-PE-Cy5 (eBioscience, San Diego, CA, USA) and negative control immunoglobulin G (IgG) were used for 30 min of staining. The cells were gated on F4/80- and CD86-positive expression, which were identified as the M1 macrophages. In addition, the cells were gated on F4/80- and CD206-positive expression, which were identified as the M2 macrophages. Unstained and fluorescein-conjugated isotypic cells served as the controls. A FACS Calibur (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) flow cytometer was employed for detection, and the data were quantified using the FlowJo software.
5
Fabrication of Nano-Hydroxyapatite/Chitosan Microspheres
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Acetic acid (Alfa Aesar, Heysham, UK) was mixed with deionized water to prepare a solution of 1% v/v acetic acid. Then, 1 g of chitosan powder (Aladdin, Shanghai, China) was dissolved in the acetic acid solution at a concentration of 1% w/v, followed by the addition of 1 g of nano-hydroxyapatite (Aladdin). The mixture was sonicated for 10 min to prepare a 1% w/v solution of nano-hydroxyapatite (nHA)/chitosan (CS). Subsequently, the nHA/CS solution was injected into a 1.0 M sodium hydroxide solution (Aladdin), using a jet apparatus comprising an electrostatic spray system (SS-X3, Yongkang Leye, Beijing, China) and a 5 mL syringe, resulting in the formation of nHA/CS microspheres. The collected microspheres were washed repeatedly with deionized water and then filtered through a 100-μm filter (Corning, New York, United States). After sterilization with 75% ethanol, the microspheres were washed with phosphate buffered saline (PBS) (Biosharp, Anhui, China) and stored in PBS solution at 4°C for future use.
6
Adipocyte Immune Cell Isolation
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After a 1-minute transcardial perfusion with PBS after sacrifice, eWAT samples were collected and digested as described previously (83 (link), 84 (link)). In short, eWAT samples were minced and incubated for 1 hour at 37°C in an incubator under agitation (60 rpm) in HEPES-buffered Krebs solution, containing 0.5–1 g/L collagenase type I from Clostridium histolyticum (Sigma-Aldrich), 2% (w/v) dialyzed BSA (fraction V; Sigma-Aldrich), and 6 mM D-Glucose (Sigma-Aldrich). The samples were passed through a 100 μm filter (Corning Life Sciences), which was washed with PBS supplemented with 2.5 mM EDTA and 5% FCS. After allowing the adipocytes to settle for approximately 10 minutes, the infranatant, consisting of immune cells, was collected and pelleted at 350g for 10 minutes at room temperature. The pellet was treated with erythrocyte lysis buffer, washed with PBS/EDTA/FCS, and counted using a hemocytometer.
7
Isolation of Lymphocytes from Tissues
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Single-cell suspensions were prepared from inguinal and mesenteric lymph nodes by mechanical disruption and passed through 100 μm filter (Corning). Colonic lamina propria T cells were isolated, as previously described48 (link). Briefly, colons were opened longitudinally and contents were flushed with ice-cold Hanks balanced salt solution, HBSS (Cellgro). Each colon was cut into small pieces and washed with HBSS solution supplemented with 5% FCS (HyClone) and 2 mM EDTA at 37 °C. A single-cell suspension was obtained after treatment with Collagenase D (1.0 mg/ml) and DNase I (0.1 mg/ml) (both from Roche). A purified and concentrated suspension of lamina propria lymphocytes was obtained after centrifugation on Percoll (GE Healthcare) gradient (45% and 70%). The interface, enriched in leukocytes, was collected and used for experiments. Lungs and liver were harvested, and lymphocytes were isolated by enzymatic digestion for 20 min, using Collagenase D (1.0 mg/ml) and DNase I (0.1 mg/ml) (both from Roche) at 37 °C. For T cell enrichment, Lymphocyte Separation Medium (Corning) was used. The interphase was collected and used for further analysis.
8
Hepatocyte Isolation via Two-Step Perfusion
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The process of isolating primary hepatocytes involved a two-step perfusion method. Initially, the liver was subjected to pre-perfusion with Hanks’ buffer, supplemented with EDTA and Hepes, by inserting the cannula through the superior vena cava and cutting the portal vein. This was followed by a low-flow perfusion with digestion buffer containing freshly added Liberase, which lasted for approximately 10 minutes. The digestion was halted using isolation buffer, and the cells were gently scraped away from the matrix using a cell scraper. Subsequently, the cell suspension was filtered through a 100-μm filter from Corning, and the hepatocytes were purified using two low-speed centrifugation steps that lasted for 2 minutes at 50 g.
9
Isolation of Immune Cells from Murine Adipose Tissue
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After a 1 minute transcardial perfusion with PBS post sacrifice, eWAT samples were collected and digested as described previously (27, 28) . In short, eWAT samples were minced and incubated for 1 hour at 37°C in an incubator under agitation (60 rpm) in HEPES-buffered Krebs solution, containing 0.5-1 g/L collagenase type I from Clostridium histolyticum (Sigma-Aldrich), 2% (w/v) dialyzed bovine serum albumin (BSA, fraction V; Sigma-Aldrich) and 6 mM D-Glucose (Sigma-Aldrich). The samples were passed through a 100 μm filter (Corning Life Sciences) which was washed with PBS supplemented with 2.5 mM EDTA and 5% FCS. After allowing the adipocytes to settle for ~10 min, the infranatant, consisting of immune cells, was collected and pelleted at 350 x g for 10 min at room temperature. The pellet was treated with erythrocyte lysis buffer, washed with PBS/EDTA/FCS, and counted using a hemocytometer.
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