ILC2s were isolated from the lung tissues according to the previous study [41 (link)]. In short, the lung tissues were filtered via a 40-µm cell strainer and placed in a six-well culture dish with 7 ml RPMI-1640 medium (31800, Solarbio, Beijing, China) containing 50 µg/ml Liberase TM (5401119001, Sigma, USA) and 1 µg/ml DNase I (BS137, Biosharp, Hefei, China). After treatment with red blood lysis (C3702, Beyotime, Shanghai, China), the samples were fractionated with Percoll (BS909, Biosharp, Hefei, China). The acquired cells were incubated with PE anti-mouse cluster of differentiation (CD)45 (clone 30-F11) antibody (F2104502, Multisciences Biotech, Hangzhou, China), FITC anti-mouse CD25 antibody (FITC-65137, Proteintech, Wuhan, China), PE anti-mouse ST2 (clone RMST2-2) antibody (12-9335-80, ThermoFisher Scientific, USA) and PE anti-mouse receptor tyrosine kinase (c-Kit) antibody (161605, Biolegend, USA) at 4 °C for 30 min in the dark. Ultimately, ILC2s were isolated and collected by gating strategies (gated as live CD45 + CD25 + ST2 + c-Kit + cells) using flow cytometry (NovoCyte, Agilent, USA).
Percoll
Manufactured by Biosharp
Sourced in China
Percoll is a colloidal silica suspension that is commonly used for density gradient centrifugation to isolate and purify cells, organelles, and other biological particles. It is a sterile, endotoxin-free solution that can be used to create density gradients with a wide range of densities.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Biosharp (2 mentions)
Liberase, by Merck Group (1 mentions)
Novocyte, by Agilent Technologies (1 mentions)
Rpmi 1640 medium, by Solarbio (1 mentions)
C3702, by Beyotime (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dextran sulfate sodium (dss), by MP Biomedicals (1 mentions)
Dispase 2, by Roche (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Collagenase 4, by Biosharp (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Fitc anti mouse cd4 antibody, by BioLegend (1 mentions)
Accutase, by Merck Group (1 mentions)
Apc anti mouse cd8a antibody, by BioLegend (1 mentions)
Pe antimouse cd3ε antibody, by BioLegend (1 mentions)
Trustain fcx plus anti mouse cd16 32, by BioLegend (1 mentions)
5 protocols using percoll
1
Isolation of Lung ILC2s by Flow Cytometry
2
Ovarian Granulosa Cell Isolation in PCOS
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This study was approved by the Ethics Committee of Renmin Hospital of Wuhan University. Ovarian GCs were collected from 20 PCOS patients and 20 healthy controls who underwent in vitro fertilization (IVF) at the Reproductive Medical Center, Renmin Hospital of Wuhan University, employing the GnRH agonist long protocol or ultralong GnRH agonist protocol. Signed informed consent was received from all subjects (No. WDRY2019-K077). The Rotterdam criteria from 2003 were used to diagnose PCOS in women, and the luteinized GCs of the patients were extracted from the follicular fluid. The follicular fluid was collected and centrifuged at 1,500 rpm for 10 min, after which the pellets were resuspended in phosphate-buffered saline (PBS). Then, GCs were separated and centrifuged at 1,800 rpm for 20 min in 50% Percoll (Biosharp, Anhui, China). The procedures for animal experiments were performed following the Guide for the Care and Use of the Animal Experiment Center Renmin Hospital of Wuhan University, and the approval ID for the animal experiment was WDRM20191203.
3
Isolation and Culture of Primary Chicken Hepatocytes
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The isolation and culture of primary chicken hepatocytes was based our previously reported procedure (Zhang et al., 2022b ). Ten 15-day-old specific pathogenic free chicken embryos (Shandong Haotai Experimental Animal Breeding Co., Shandong, China) were sterilized and dissected, and the livers were pooled, and cut into small pieces (1 mm × 1 mm), then digested by using 0.25% trypsin-EDTA (Gibco, New York) at 37°C for 30 min. The hepatocytes collected via low-speed centrifugation (1,000 r/min × 5 min), then further purified by centrifugation through 60% Percoll (60% v/v, Biosharp, Anhui, China). Subsequently, primary chicken embryonic hepatocytes at 2 × 105 cells/mL were seeded in 6-well plates with Dulbecco's modified Eagle medium (DMEM , Gibco, New York) in a humidified atmosphere containing 95% air and 5% CO2 at 37°C.
4
Wogonin Dissolution and Cell Preparation
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Wogonin (purity >98%) purchased from Nanjing Zelang Medical Technology (Nanjing, Jiangsu, China) was dissolved in 1 M NaOH as a stock solution, stored at −20°C, and freshly diluted with RPMI 1640 medium to the final concentration. The working solution of NaOH was less than 0.1 μM. DSS (molecular weight: 36,000–50,000) was obtained from MP Biomedical (Solon, Ohio, USA). Lymphoprep was obtained from Axis‐shield (Oslo, Norway). Collagenase IV, Dnase I and Percoll were purchased from BIOSHARP (Hefei, Anhui, China). Dispase II was obtained from Roche (Basel, Switzerland).
5
Isolation and Analysis of Hippocampal Immune Cells
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Animals were transcardially perfused with precooled PBS. Then, the whole brain was removed and placed in precooled complete DMEM to further isolate the hippocampus under a stereomicroscope. The hippocampi were scissor-minced and manually homogenized. The tissue was subsequently dissociated by adding accutase (Sigma-Aldrich) and incubated at 37 °C, 5% CO2 in a cell culture incubator for 15 min. The reaction was stopped by adding serum-containing culture medium. Single-cell suspensions were filtered with a 70-µm nylon mesh. Density gradient centrifugation using Percoll (Biosharp) was performed to isolate the mononucleated immune cells. Samples were washed with PBS three times. Fc receptors were blocked by TruStain FcX™ PLUS (anti-mouse CD16/32, Biolegend) for 5 min on ice. PE anti-mouse CD3ε antibody (Biolegend), FITC anti-mouse CD4 antibody (Biolegend) and APC anti-mouse CD8a antibody (Biolegend) were added for 20 min at 4 °C and protected from light. Isotype-matched antibodies were used as controls. After cleaning the cells with PBS three times, samples were run on a BD LSRFortessa cell analyzer.
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