Mice were sacrificed, peripheral blood (PB) was collected, liver, spleen (SP), and mesenteric lymph nodes (LN) were removed. Spleen, and MLN were mechanically dissociated and processed through a 100-um cell strainer (BD Falcon), and suspended in HBSS. Lymphocytes were isolated by Ficoll-Hypaque (DAKEWE, SZ, China) density gradient centrifugation from cell solution and diluted in blood solution. Isolated cells were washed twice in HBSS and re-suspended at 2 × 106 cells/ml in complete RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 50 μM 2-mercaptoethanol.
Ficoll hypaque
Manufactured by Dakewe
Sourced in China
Ficoll-Hypaque is a density gradient medium used for the isolation and purification of cells, particularly mononuclear cells, from whole blood or other biological samples. It is a mixture of Ficoll, a synthetic polymer, and sodium diatrizoate, a radiographic contrast agent. The core function of Ficoll-Hypaque is to separate different cell types based on their density during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Liver dissociation kit, by Miltenyi Biotec (2 mentions)
Collagenase 4 dnase 1 mix, by Thermo Fisher Scientific (2 mentions)
100 um cell strainer, by BD (1 mentions)
Clone 1a8, by BioLegend (1 mentions)
Clone hk1, by BioLegend (1 mentions)
Clone bm8, by BioLegend (1 mentions)
Clone m1 70, by BioLegend (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
100 μm cell strainer, by BD (1 mentions)
8 protocols using ficoll hypaque
1
Isolation of Lymphocytes from Murine Tissues
2
Tumor-Infiltrating Lymphocyte Analysis
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The significant differences in tumor volume between IDO-APT treated group and Scr-APT treated group began to appear Mice were euthanized 2 days after the third treatment and the tumor tissue was collected. Tumor tissues were cut into small pieces and digested with collagenase D and DNase I for 30 min at 37°C. After grinding and passing through the 200 meshes strainer, TILs were isolated from the dissociated cells by Ficoll-Hypaque (Dakewe Biotech). For intracellular cytokine analysis, cells were re-stimulated with 100 ng/mL PMA and 500 ng/mL ionomycin in the presence of protein transport inhibitor cocktail for 5 h. After staining with anti-CD45-PE-Cy7 and anti-CD8-FITC, cells were fixed and permeabilized and then stained with anti-TNF-α-PE or anti-IFN-γ-APC. For Treg cells analysis, cells were stained with anti-CD45-PE-Cy7, anti-CD4-FITC, anti-CD25-PE, and anti-Foxp3-APC. For tumor-associated macrophages analysis, cells were stained with anti-CD45-PEcy7, anti-CD11b-FITC (Cat 101205, Clone M1/70, Biolegend), and anti-F4/80-APC (Cat 123115, Clone BM8, Biolegend). For granulocytic or monocytic MDSC analysis, cells were stained with anti-CD45-PEcy7, anti-CD11b-FITC, anti-Ly6C-PE (Cat 128007, Clone HK1.4, Biolegend) or anti-Ly6G-PerCP (Cat 127653, Clone 1A8, Biolegend). The stained cells were then measured by flow cytometry.
3
Isolating Menstrual Blood-Derived Stem Cells
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MenSCs were provided by the Innovative Precision Medicine (IPM) Group, and the specific process of obtaining cells was as above [37 (link), 47 (link), 48 (link)]. In brief, menstrual blood samples were collected from healthy young women (n = 3) aged 20 to 30 years during menstruation using Divacup (Kitchener, ON). Fresh menstrual blood samples should not be stored for more than 72 hours in a storage solution containing kanamycin sulfate, cefadroxil, vancomycin hydrochloride, amphotericin B, gentamicin sulfate, and heparin in a 4°C refrigerator. Stem cells in menstrual blood were obtained by density gradient centrifugation with Ficoll-Hypaque (DAKEWE, China). The isolated interlayer cells were cultured in Minimum Essential Medium α (MEMα) (Gibco, USA) adding 15% Australian fetal bovine serum (FBS). MenSCs were completely digested with 0.25% trypsin-EDTA (Fisher Scientific, USA) for 5 min, then neutralized with complete medium, and centrifuged to complete subculture. Passages 5-8 (p5-p8) of MenSCs can be used for collection of small EVs.
4
Isolation of Liver Immune Cells
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Mice were sacrificed, and the livers were digested with a LIVER dissociation kit (Miltenyi Biotec, Germany) and dissociated into a cell suspension. Then, immune cells were isolated by Ficoll-Hypaque (DAKEWE, SZ, China) density gradient centrifugation from the cell solution. Isolated cells were washed twice in HBSS and resuspended at 2 × 106 cells/ml in complete RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 50 µM 2-mercaptoethanol.
5
Isolation and Analysis of Primary AML Samples
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Methods used in this study were approved by the Ethics Committee of the West China Hospital. Informed consent for the use of human AML and normal peripheral blood (PB) samples for the present study was obtained from all patients according to the Declaration of Helsinki. Primary AML samples evaluated in this study were taken from six AML patients (sample #1, #2, #3: FLT3-ITD positive; sample #4, #5, #6: FLT3-ITD negative), and normal PB samples were taken from three normal volunteers (sample #7, #8, #9). PBMCs were isolated using density gradient centrifugation with Ficoll-Hypaque (DAKEWE) within 24 h of collection.
6
Isolation and Analysis of Murine Liver Cells
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Mice were perfused with sterile saline to remove blood from the body before obtaining the liver tissue. Analysis of livers cells was performed on cell suspensions obtained from organ homogenates that were digested with a LIVER dissociation kit (Miltenyi Biotec, Germany). Non-parenchymal liver cells were isolated by Ficoll-Hypaque (DAKEWE, SZ, China) density gradient centrifugation from the cell solution [11 (link)]. They were washed in HBSS and resuspended at 2 × 106 cells/ml in complete RPMI 1640 medium. The number of cells was measured under a microscope (Olympus, Shinjuku, Tokyo, Japan).
7
Isolation of Immune Cells from Malaria-Infected Mice
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Mice were sacrificed at different time points after malaria infection. The liver, lung, blood, spleen, and peripheral blood mononuclear cell (PBMC) were collected, firstly. Then lung was cut to small pieces and incubated in 5 ml of digestion buffer (collagenase IV/DNase I mix, Invitrogen Corporation) for 30 min at 37 °C. The digested lung tissue was pressed through 200-gauge stainless-steel mesh, and then was suspended in Hank’s balanced salt solution (HBSS). Liver, lung, spleen, and mesenteric lymph nodes (MLN) were mechanically dissociated and processed through a 100-μm cell strainer (BD Falcon), and suspended in HBSS. Lymphocytes were isolated by Ficoll-Hypaque (DAKEWE) density gradient centrifugation. Isolated cells were washed twice in HBSS and re-suspended at 2×106 cells/ml in complete RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 50 μM 2-mercaptoethanol.
8
Isolation of Lung Lymphocytes in Mice
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Mice were anesthetized and fixed from wk 5 and 7 after infection. The excised
lung was cut to small pieces and incubated in 5 mL of digestion buffer
(collagenase IV/DNase I mix, Invitrogen Corporation) for 30 min, at 37 and 5%
carbon dioxide. The digested lung tissue was pressed through 200-gauge
stainless-steel mesh, and then was suspended in Hank’s balanced salt solution
(HBSS). Lymphocytes were isolated by Ficoll-Hypaque (DAKEWE, China) density
gradient centrifugation. Isolated cells were washed twice in HBSS and
re-suspended in complete RPMI 1640 medium supplemented.
lung was cut to small pieces and incubated in 5 mL of digestion buffer
(collagenase IV/DNase I mix, Invitrogen Corporation) for 30 min, at 37 and 5%
carbon dioxide. The digested lung tissue was pressed through 200-gauge
stainless-steel mesh, and then was suspended in Hank’s balanced salt solution
(HBSS). Lymphocytes were isolated by Ficoll-Hypaque (DAKEWE, China) density
gradient centrifugation. Isolated cells were washed twice in HBSS and
re-suspended in complete RPMI 1640 medium supplemented.
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