Blood was taken from either the left or the right caudal vein with 1 ml syringe and rapidly transferred to heparin saline anticoagulant tubes. For each rat, 0.5–1 ml of venous blood was harvested. Spleen single-cell suspensions were obtained by grinding followed by filtration through a nylon mesh. For leukocyte retrieval, the samples from the blood and spleen were centrifuged at 3000 rpm for 4 min at 4 °C. The pellets were treated with the red blood cell lysis buffer (Beyotime Biotechnology Co. Ltd., Jiangsu, China) and washed twice with phosphate buffer solution (PBS, HyClone Laboratories Inc., Logan, UT) to remove red blood cells. For brain cell isolation, the contralateral and ipsilateral hemispheres were cut into small pieces, ground, and filtered through a 70-μm cell strainer (Miltenyi Biotec, Bergisch Gladbach, Germany) to obtain a single cell suspension. Cell pellets were resuspended in 70% Percoll (Solarbio, Beijing, China), transferred into 15 ml tubes, and then overlaid with 30% Percoll. The mononuclear cells were harvested after centrifuging at 2000 rpm for 25 min at room temperature [25 (link)]. The leukocytes were analyzed for membrane marker expression by flow cytometry.
Percoll
Manufactured by Solarbio
Sourced in China, United States
Percoll is a colloidal silica suspension used for cell separation and gradient centrifugation. It is designed to create density gradients for the isolation and purification of cells, organelles, and other biological particles.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Dnase 1, by Merck Group (6 mentions)
Penicillin, by Solarbio (3 mentions)
Streptomycin, by Solarbio (3 mentions)
Soluble anti cd28, by Thermo Fisher Scientific (3 mentions)
Anti cd3, by Thermo Fisher Scientific (3 mentions)
Collagenase 4, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
L 15 medium, by Thermo Fisher Scientific (3 mentions)
Ethylenediaminetetraacetic acid, by Thermo Fisher Scientific (2 mentions)
L dmem f12, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by MedChemExpress (2 mentions)
Phorbol myristate acetate (pma), by MedChemExpress (2 mentions)
Sytox green, by Keygen Biotech (2 mentions)
Collagenase, by Merck Group (2 mentions)
Collagenase c2139, by Merck Group (2 mentions)
Collagenase 4, by Solarbio (2 mentions)
Ionomycin ion, by Merck Group (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Dnase 1, by Roche (2 mentions)
42 protocols using percoll
1
Isolation of Immune Cells from Rat Blood, Spleen, and Brain
2
Isolation and Characterization of Immune Cells from Tumor Tissues
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Tumor tissues were cut and placed in DMEM solution containing 1 mg/ml collagenase IV (Solarbio, China) and in a 37°C water bath for 1 h. After digestion, the cells were placed on ice, filtered through a 100 mesh sieve(150μm), centrifuged, resuspended with 40% Percoll (Solarbio, China) and placed on the surface of 80% Percoll (Solarbio, China), and immune cells were obtained by density gradient centrifugation. The above immune cells, human kidney cancer cell lines and Jurkat cells were washed using PBS buffer and treated under dark conditions with antibodies for flow cytometry. All samples were analyzed using a Beckman Coulter CytoFLEX S flow cytometer, and FlowJo software was used to analyze the data.
3
Isolation and Culture of Human BMSCs
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Primary human BMSCs were isolated from the iliac crest BM aspirates of healthy donors after informed consent was obtained. The clinical sample collections complied with guidelines in the Declaration of Helsinki and were approved by Nanjing Drum Tower Hospital's institutional review board. All patients had signed the consent forms. BM mononuclear cells were isolated by Percoll (Solarbio), Dulbecco's modified Eagle's medium (DMEM) (Euroclone) with 1,000 mg/ml glucose and L−glutamine, centrifuged and plated at a density of 1,000 cells/cm2. BMSCs were cultured in L-DMEM/F12 (GIBCO, USA) with 15% fetal bovine serum (GIBCO) at 37°C with 5% CO2. Four days later, non-adherent cells were removed carefully and the culture medium was refreshed. When primary cultures became almost confluent, the culture was treated with 0.5 ml of 0.25% trypsin containing 0.02% mmol/L ethylenediamine tetraacetic acid (GIBCO, USA) for 4 min at room temperature (25°C). A purified population of BMSCs was obtained 1 week after the initiation of culture.
4
Spinal Cord Injury Single-Cell Analysis
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Rats were anesthetized with 10% trichloroacetaldehyde and then perfused with PBS after 7 days of SCI. Then, 0.5-cm spinal cords, including the injury epicenter or the same segments for the sham group, were removed. The spinal cords were then grounded and centrifuged to obtain a single-cell suspension. Then, mononuclear cells were isolated by Percoll (Solarbio) gradient centrifugation. Briefly, 70% Percoll solution, 30% Percoll solution, and cell suspensions were added to a 15 ml conical tube in sequence, the cells were centrifuged at 300 g for 30 min at 20°C. After centrifugation, the cell debris layer was discarded and the rest parts were washed two times before their use. Then, the primary antibodies were incubated with the cells for 30 min; after that, the cells were washed with PBS and then fixed with 0.5 ml of 1% PFA (MACKLIN); finally, they were detected by BD FACSVerse flow cytometer (BD Bioscience). We used isotype control antibodies to estimate the non-specific staining that was subtracted from the specific staining results. Supplementary Table 3 shows the details of the antibodies. We use FlowJo 7.6 software (FlowJo) to analyze the data.
5
Isolation and Characterization of Liver Mononuclear Cells
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The liver mononuclear cells were collected and suspended in PBS as described previously (18 (link)). In general, the liver cell suspension was centrifuged at 30 g for 5 min. Supernatants were collected, washed in PBS, and resuspended in 40% Percoll (Solarbio, Beijing, China). Furthermore, the cell suspension was gently overlaid onto 70% Percoll and centrifuged for 20 min at 800 g. Liver mononuclear cells were collected from the interphase, washed twice in PBS, and resuspended in PBS for FACS analysis. The expression of cell surface molecules was detected by staining with antibodies against CD3 (APC-Cy7, Biolegend, San Diego, USA), CD4 (FITC, Biolegend, San Diego, USA), CD8 (Percp-Cy5.5, Biolegend, San Diego, USA), CD25 (PE, Biolegend, San Diego, USA), CD69 (APC, Biolegend, San Diego, USA), and DAPI (Biolegend, San Diego, USA), diluting each antibody according to the manufacture’s instruction.
6
Heart-Infiltrating Cell Isolation
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The heart-infiltrating cells were isolated, as previously described (18 (link)). Briefly, the heart tissues were obtained and cut into pieces after which the tissue fragments were digested with 1 mg/mL of collagenase B (Roche 11088815001) or Collagenase Type 2 (Sangon Biotech A004174-0100) in HEPES buffer then rotated gently for 45 min at 37°C. In addition, cell suspensions were filtered with 70-μm cell strainers (BIOFIL) and Percoll (Beijing Solarbio Science & Technology Co., Ltd.) was used to purify heart mononuclear cells through density centrifugation.
7
Isolation and RNA extraction from cMPNs
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A total of 12 samples (six JAK2V617F-positive cMPNs patients and six normal controls) were recruited from Tongji hospital of Tongji University (Table S1, shown in Additional file 1 ). The diagnosis of cMPNs was defined according to World Health Organization (WHO) criteria [8 (link)]. The use of clinical samples in our study was according to the Declaration of Helsinki and was approved by the Medical ethic committee of Tongji hospital of Tongji University on Feb. 2021 (Number: 2021-KYSB-177). Written informed consent was obtained from each participant. Mononuclear cells are isolated from bone marrow using density gradient separation (Percoll, Solarbio life sciences, Beijing, China). Total RNA was extracted from bone marrow mononuclear cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instruction. Total RNA from each sample was quantified by the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
8
Isolation and Quantification of NETosis
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Spleen neutrophils were isolated with Percoll (Solarbio, Beijing, China) gradients as described (23 (link)). NETs assay was performed as protocols published (24 (link)). Neutrophils were resuspended in RPMI-1640 containing 5-10% FBS and plated at 5×105 cells per well in 24-well plates (Thermo Fisher, Massachusetts, US). After incubation for 0.5-1 h, the cells were stimulated with PMA (8 μM, MedChemExpress, New Jersey, USA), ionomycin (10 μM, MedChemExpress, New Jersey, USA) or fecal microbiota (2×107 CFU), and the neutrophils were incubated for an additional 3 h to form NETs. Cells were stained with SYTOX green (1:1000, KeyGEN BioTech, Nanjing, China) for NETs quantification or, in some experiments, fixed in 2% paraformaldehyde, permeabilized, blocked, and stained with anti-H3cit (1:1000, Abcam, cat. no. ab5103) and anti-rabbit secondary antibodies (1:200, Servicebio, Wuhan, China). Images were acquired on an Axiovert 200 M wide-field fluorescence microscope (Nikon) with a coupled camera. The percentages of H3cit high cells and NETs were determined from five or six nonoverlapping fields per well, and the average was taken from 2-3 biological repetitions in every experiment.
9
Mitochondrial Isolation and Protein Quantification
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The process was according to the previously described [32] (link) with modifications. Samples were placed in a 9× volume of ice-cold mitochondria isolation buffer (mmol/L: mannitol 225, sucrose 75, K2HPO4 2, EGTA 1, pH 7.2) and homogenized until no visible particles. The homogenate was centrifuged at 1300×g, 5 min, 4 °C (Eppendorf, Germany) and the supernatant was then layered on 15% (vol/vol) Percoll (Solarbio) and subjected to a centrifugation at 36,500×g, 14 min, 4 °C (HITACHI, Japan). Decant the supernatant, re-suspend the pellet with mitochondria isolation buffer and centrifuge at 10,000×g, 10 min, 4 °C. Decant the supernatant, re-suspend the pellet with mitochondria isolation buffer and centrifuge at 8000×g, 10 min, 4 °C.
Mitochondrial protein concentration was determined at 540 nm by NanoDrop 2000c (Thermo Scientific).
Mitochondrial protein concentration was determined at 540 nm by NanoDrop 2000c (Thermo Scientific).
10
Isolation and Separation of PBMC Subsets
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Approximately 5 mL of diluted peripheral blood (heparinized) was further diluted with Hank's buffer at a ratio of 1 : 3 and was slowly added to 5 mL lymphocyte separation medium (Percoll, Solarbio, Beijing) in a 15 mL centrifuge tube. The mononuclear leukocyte layer was isolated by centrifugation at 1200 rpm for 20 min. Cells at the interface were collected and slowly washed with 5 mL of sterile Hank's buffer and centrifuged at 1000 rpm for 5 min. Then, the cell pellets were washed and centrifuged 2-3 times with Hank's buffer using the same parameters. After that, magnetic beads were used to separate the CD4+ T cells from the CD19+ B cells. Total PBMCs from MG patients, PBMCs from healthy controls, CD4+ T cells from MG patients, and CD19+ B cells from MG patients were resuspended in DMEM complete medium (HyClone, USA) with 15% fetal bovine serum (Gibco, USA). The cell suspension was seeded in a culture flask (Corning, USA).
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