Mononuclear cells in the brain of pre-treated, CT2A-inoculated mice were isolated 7, 14, 21 or 28 days post tumor inoculation as previously described31 (link). Briefly, isolated brains were digested in Collagenase D (Roche, Anderlecht, Belgium) and DNase (Invitrogen), and filtered prior to Percoll gradient centrifugation (Sigma). After recovering the mononuclear cell interphase, assessment of the immune cells was done by flow cytometry. Therefore surface staining for T cell markers was performed following zombie yellow viability staining (Table 1 ). Foxp3-PE staining kit (eBioscience, Vienna, Austria) was used according to the manufacturer’s protocol to detect intracellular Foxp3. For the intracellular IFN-γ staining, cells were stimulated for 3 hours in vitro with 100 ng/mL phorbol myristate acetate, 1 mg/mL ionomycin and 0.7 mg/mL monensin (all from Sigma-Aldrich). After viability and surface staining cells were permeabilized with a buffer containing 0.5% saponin and 0.5% bovine serum albumin prior to IFN-γ staining.
Percoll gradient centrifugation
Manufactured by Merck Group
Sourced in Belgium
Percoll gradient centrifugation is a laboratory technique used to separate and purify cells, organelles, or other biological particles based on their density. It involves creating a density gradient using the colloidal silica polymer Percoll, which allows the separation of different components within a sample during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase d, by Roche (4 mentions)
Dithiothreitol (dtt), by Merck Group (3 mentions)
Dnase, by Thermo Fisher Scientific (2 mentions)
Facsaria 3 cell sorter, by BD (2 mentions)
Dispase, by BD (2 mentions)
Dnasea, by Merck Group (2 mentions)
Fastprep 24 instrument, by MP Biomedicals (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Corning (2 mentions)
Hanks balanced salt solution (hbss), by Merck Group (2 mentions)
Collagenase 2, by Merck Group (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Collagenase 3, by Worthington (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Monensin, by Merck Group (1 mentions)
100 μm strainer, by BD (1 mentions)
10 protocols using percoll gradient centrifugation
1
Isolation and Analysis of Brain Immune Cells in Murine Glioma
2
Isolation of Primary Murine Hepatocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary hepatocytes were isolated from WT and TLR4-/- mice by a two-step collagenase perfusion technique as previously described [23 (link)]. After preperfusion with GBSS solution through the portal vein, the liver was perfused with 50 mL of GBSS solution containing 0.05% collagenase and cut into small pieces to isolate hepatocytes. Hepatocytes were purified by a series of low-speed (1’×50 g) centrifugation steps and Percoll gradient centrifugation (50% v/v, Sigma). Purified hepatocytes were treated under 3D culture conditions.
3
Isolation and Purification of Intestinal Epithelial and Lamina Propria Leukocytes
4
Isolation of Primary Murine Hepatocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Male C57/B6J and Nrf2−/− mice (8 weeks old) were used for isolating primary hepatocytes. Hepatocytes were isolated by a two-step collagenase perfusion technique. Briefly, the inferior vena cava was cannulated with angiocatheter and the portal vein was cut. The liver was perfused via the inferior vena cava with 100 mL of PBS at 37°C, followed by perfusion with 100 mL of collagenase type IV (Wellington) in HBSS containing Ca2+ and Mg2+ (GIBCO). After the liver was digested, it was dissected and cut into small pieces and passed through a 100 μm strainer (Falcon). Hepatocytes were separated from non-parenchymal cells by low-speed centrifugation and further purified by Percoll gradient centrifugation (50% v/v, Sigma). Cells were plated at a density of 0.3 × 106 on a 6-well collagen-coated plate. Hepatocytes were allowed to recover overnight and experiments were started 24 h post isolation.
5
Isolation of Immune Cells from Murine Tissues
6
Isolation of Mucosal Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small intestine was removed, cleaned from remaining fat tissue and washed in ice-cold PBS (Corning). Peyer’s patches were identified and eliminated. Small intestine was opened longitudinally and washed in ice-cold PBS. Dissociation of epithelial cells was performed by incubation on a shaker at 37°C in HBSS (Sigma-Aldrich) containing 10 mM Hepes and 5 mM EDTA (both Thermo Fisher Scientific) or 1mM DTT (Sigma-Aldrich) two times for 15 min. After each step, samples were vortexed and the epithelial fraction discarded. Afterwards, remaining tissue was chopped into small pieces and enzymatic digestion was performed using Dispase (5 U/ml; Thermo Fisher Scientific), Collagenase III (1 mg/ml; Worthington) and DNaseI (20 μg/ml; Sigma-Aldrich). Leukocytes were further enriched by a 40%/80% Percoll gradient centrifugation (Sigma-Aldrich).
One PBS-perfused lung lobe was minced and incubated using same conditions and enzymatic cocktail as for the intestine for 45 min at 37°C. Afterwards lung was vortexed, mashed through a 70 μM cell strainer and purified over a 40%/80% Percoll gradient.
Mesenteric lymph nodes were chopped and incubated in RPMI medium supplemented with 1% BSA (Sigma-Aldrich), DNaseI (20 μg/ml) and Collagenase II (1 mg/ml; Sigma-Aldrich) for 20 min at 37°C. Specimens were mechanically dissociated and filtered through a 70 μM cell strainer.
One PBS-perfused lung lobe was minced and incubated using same conditions and enzymatic cocktail as for the intestine for 45 min at 37°C. Afterwards lung was vortexed, mashed through a 70 μM cell strainer and purified over a 40%/80% Percoll gradient.
Mesenteric lymph nodes were chopped and incubated in RPMI medium supplemented with 1% BSA (Sigma-Aldrich), DNaseI (20 μg/ml) and Collagenase II (1 mg/ml; Sigma-Aldrich) for 20 min at 37°C. Specimens were mechanically dissociated and filtered through a 70 μM cell strainer.
7
Multiparameter Flow Cytometry Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometric analysis was performed as described earlier12 (link). In brief, animals were sacrificed by lethal injection of Nembutal at the indicated time points, and perfused with PBS (Lonza, Belgium). Single cell suspensions were obtained after mincing with scalpels and 30′ incubation with DNase (Invitrogen) and CollagenaseD (Roche). Mononuclear cells were separated from debris via Percoll gradient centrifugation (Sigma), and the intermittent layer was washed twice with PBS. Surface stainings were performed with antibodies as mentioned in Table 1 .
Flow cytometry antibodies.
| Antigen | Fluorochrome | Origin |
|---|---|---|
| CD45 | AF700 | Ebioscience |
| CD11b | BV421 | BD |
| Ly6C | AF647 | Biorad |
| Ly6G | FITC | BD |
| MHCII | PerCP Cy5.5 | Biolegend |
| Mannose Receptor | PE | Biolegend |
| Isotype Rat IgG2a,k | PE | Biolegend |
| Live/dead | Zombie Yellow | Biolegend |
| CD3 | FITC/PE | Ebioscience |
| CD4 | PerCP Cy5.5/APC-eF780 | Ebioscience |
| CD8 | BV421 | BD |
| NKp46 | APC | Biolegend |
| FoxP3 | PE | Ebioscience |
| PD-1 | PE | BD |
| IFN-γ | PerCP Cy5.5 | BD |
8
Isolation and Culture of Human GBM Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All Human samples were collected with written informed consent under a protocol approved by the Institutional Review Board of the Samsung Medical Center (2010–04–004, Seoul, Korea). Parts of the surgical samples were enzymatically dissociated, and then red blood cells were removed by percoll gradient centrifugation (Sigma-Aldrich). Dissociated cells were maintained in the ‘NBE’ conditions consisting of Neuro-Basal Media, N2 and B27 supplements (×1/2 each), 2 mM L-glutamine, 100U/ml penicillin and streptomycin (Invitrogen), and human recombinant EGF and bFGF (50 ng/ml each; R&D Systems). The human GBM cell line U-87 MG (ATCC) was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum, 100U/ml penicillin and streptomycin (Invitrogen).
9
Isolation and Purification of Intestinal Epithelial and Lamina Propria Leukocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of lamina propria leukocytes was performed as described previously61 . In brief, intestines were isolated, cut open longitudinally and washed briefly in PBS. Dissociation of epithelial cells was performed by shaking at 100 rpm/min at 37°C in HBSS containing 5 mM EDTA and 10 mM Hepes for 20 min. Epithelial cells were collected and spun down for 5 min at 2000 rpm/min at 4°C and cell pellets were resuspended in Trizol using a FastPrep®-24 Instrument (MP Biomedicals). To obtain highly purified epithelial cells, the cell pellets were digested for 20 min using Dispase (5 U/ml; BD), Collagenase D (0.5 mg/ml; Roche) and DNaseA (0.5 mg/ml; Sigma-Aldrich). Cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI), CD45 and EpCAM antibodies and sorted using a BD FACSAria III cell sorter (BD Biosciences). The remaining lamina propria fraction was cut into pieces of 1 mm2 before performing enzymatic digestion using the same enzyme mix as for the digestion of epithelial cells. Lymphocyte enrichment was performed by Percoll gradient centrifugation (Sigma-Aldrich).
10
Isolation of Mucosal Immune Cells
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!