Mixed glial cultures containing astrocytes, pericytes, endothelial cells, and microglia were isolated from human brain tissue as described previously [47 (link)] and used at passage two. Isolated pericyte cultures were generated from these initial mixed glial cultures by subsequent passaging in order to dilute out non-proliferating microglia, astrocytes, and endothelial cells as described previously [48 (link)]. Isolated microglial cultures were generated as described previously [49 (link)]. Cells were harvested using 0.25% trypsin- 1 mM EDTA (Gibco, CA, USA) with mixed glial cultures and microglia cultures also utilising gentle detachment with a cell scraper (Falcon, MA, USA) due to strong microglial attachment. Viability was determined by trypan blue exclusion (Gibco). Mixed glial and pericyte cultures were plated at 15,000 cells/cm2 and isolated microglia were plated at 30,000 cells/cm2 in Nunc™ microwell plates with Nunclon™ Delta surface (Nunc, Denmark). All cultures were maintained in DMEM/F12 (Gibco), 10% fetal bovine serum (FBS; Moregate, Australia) and 1% penicillin streptomycin glutamine (PSG; Gibco).
Cell scraper
Manufactured by BD
Sourced in United States
A cell scraper is a laboratory instrument used to detach adherent cells from the surface of a cell culture vessel, such as a petri dish or tissue culture flask. It consists of a handle and a flat, flexible blade that is gently scraped across the vessel's surface to dislodge the cells.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
24 well plate, by Corning (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Trypan blue exclusion, by Thermo Fisher Scientific (1 mentions)
Trypsin 1 mm edta, by Thermo Fisher Scientific (1 mentions)
Nunc microwell plate, by Thermo Fisher Scientific (1 mentions)
Nunclon delta surface, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Mcf 7 cell, by American Type Culture Collection (1 mentions)
Transfer buffer, by Boston BioProducts (1 mentions)
Methanol, by BD (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Lds buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Lympholyte m cell separation media, by Cedarlane (1 mentions)
Trypsin edta solution, by Corning (1 mentions)
Trypan blue staining solution, by Thermo Fisher Scientific (1 mentions)
Fluorobrite, by Harvard Bioscience (1 mentions)
18 protocols using cell scraper
1
Isolation and Culture of Mixed Glial Cells
2
Single-cell PAGE of MCF-7 Cells
3
Isolation and Characterization of Legionella-Containing Vacuoles
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Differentiated U937 cells (3 × 106) were seeded onto four wells of a 6-well plate and allowed to adhere. The cells were infected as described above. After 16 h, the wells were washed in 2 ml of PBS and gently scraped with a cell scraper (Falcon) to remove cells. Free LCVs were prepared as previously described (36 (link), 69 (link)). The cells were pelleted at 233 × g for 5 min at 4°C and resuspended in 1 ml of ice-cold homogenization hypo-osmotic buffer (20 mM HEPES–KOH, pH 7.2, 250 mM sucrose, 5 mM EGTA) with cOmplete protease inhibitor cocktail. The cells were then added to a type B Dounce homogenizer (Kimble Chase) and subjected to Dounce homogenization 5 times. Lysis of >90% of the cells was verified by trypan blue exclusion assay (101 (link)). The LCVs were separated from intact host cells and nuclei by centrifugation at 524 × g for 3 min at 4°C. The supernatant (500 μl) was centrifuged at 1,455 × g for 5 min onto poly-l -lysine-coated coverslips in a 24-well plate. The plate was incubated for 15 min at 37°C in a buffered, humidified chamber to help facilitate adhesion. The free LCVs were then fixed in 4% paraformaldehyde and analyzed by indirect immunofluorescence assay and confocal microscopy as detailed above.
4
Hippocampal and Subcutaneous Stem Cell Injection
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All animal housing conditions, surgical procedures, and postoperative care were approved by and conducted according to the Institutional Animal Care and Use Committee (IACUC) guidelines at the University of California, Irvine. All hESC and hNSC preparations took place during the day of transplantation/injection. All cells were maintained under normal culture conditions prior to harvesting and collection for hippocampal/subcutaneous injection. For hippocampal hNSC injections, cells were dissociated to single cells using TrypLE and resuspended in X-Vivo 15 to yield 7.5 × 104 cells/μl. hESCs were harvested in a similar manner for hippocampal injection but care was taken to avoid single-cell dissociation by using a cell scraper (BD Falcon) rather than TrypLE. Hippocampal cell preparations were kept at room temperature.
For subcutaneous leg injections of both hESC and hNSC, cells were prepared similarly to hippocampal preparations but were resuspended in Matrigel rather than X-Vivo 15 and kept on ice to prevent Matrigel from polymerizing prematurely. Dilutions ranged from 1.25 to 2.5 × 106 cells per 50 μl.
For subcutaneous leg injections of both hESC and hNSC, cells were prepared similarly to hippocampal preparations but were resuspended in Matrigel rather than X-Vivo 15 and kept on ice to prevent Matrigel from polymerizing prematurely. Dilutions ranged from 1.25 to 2.5 × 106 cells per 50 μl.
5
Exosome Protein Analysis in PC12 Cells
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Following 7 days of exosome treatment, cells were removed from the culture plate using a cell scraper (Falcon) and lysed with cell lysis buffer containing 1% Triton X-100 and a protease inhibitor cocktail (Sigma-Aldrich). After determination of the protein concentration by BCA assay, the protein samples were denatured at 90°C for 5 min. The samples (30 μg) were loaded with LDS buffer (Life technologies) in a Bis-Tris gel (Life technologies). The gel was run in MES Buffer (Boston BioProducts, Ashland, MA) at 170 V for 45 min. The proteins were then transferred to a PVDF membrane (Life Technologies) at 30 V for 2 h in the transfer buffer (Boston BioProducts) with 20% methanol (BDH, Radnor, PA). After blocking with 5% dry milk in PBS, the membrane was incubated with primary antibodies against MAP2, NSE, and glyceraldehyde phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, MA), and then the secondary antibody conjugated with horseradish peroxidase (HRP) (Life Technologies). The blots were detected with enhanced chemiluminescence (ECL) using an imager (Syngene, Frederick, MD). Western blotting of PC12 exosomes was performed using the same method described above.
6
Murine Bone Marrow-Derived Macrophage Isolation
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Murine bone marrow-derived macrophages (BMDMs) were obtained from femora and tibiae of female 6–8 week-old C57BL/6 mice, or from mice of the indicated genotypes. Macrophages were cultured in RPMI 1640 medium supplemented with 15% fetal bovine serum (Serana), 20% L-cell-conditioned medium (containing macrophage colony-stimulating factor), and 100 U/mL of penicillin-streptomycin (Sigma) in bacteriological dishes for 7 days, at 37°C + 5% CO2. For infections, BMDMs were gently scraped from plates using a cell scraper (BD Falcon) and washed three times in PBS, before seeding into tissue culture-treated plates.
7
Hippocampal and Subcutaneous Stem Cell Injection
8
In Vivo and In Vitro RBC Phagocytosis Assays
9
Cytotoxicity Evaluation of Degradable Particles
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The toxicity of the particles and their degradation products on J774A.1 is investigated by trypan blue staining. In all, 3 × 104 cells are seeded in 24-well plates (Corning) and cultured in Fluorobrite (Biochrom) + 10% FBS (Invitrogen) + 1% penicillin/streptomycin (Invitrogen) and stimulated by adding 1 µg/ml LPS (Sigma Aldrich) for 1 h. Cells incubated with cell culture medium without LPS served as negative controls. Degradable and non-degradable particles are diluted in cell culture medium and added to the cells with a concentration of either 5 or 100 µg/ml and the cells are incubated for 4, 12, 24, and 48 h. Three samples per condition are analyzed. After incubation, the cells are removed with a cell scraper (Falcon) and transferred to eppendorf tubes. The cell suspension is centrifuged and the supernatant is removed. The cell pellet was dissolved in 50 µl cell culture medium. Twenty-five microliters of the cell suspension is mixed with 25 µl Trypan blue staining solution (Gibco). Trypan blue positive cells (blue cells) were counted using Cedex XS cell counter (Innovates AG). The percentage of positive cells as a function of the total cell number was calculated.
10
Transient Transfection and HA Immunoblot
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HEK293 cells were transiently transfected with empty vector (pIRES2-EGFP) and BlTLRHA plasmid as described above. Cells were lysed in 200 μl cell lysis buffer (250 mM sacarose, 150 mM Tris, 5 mM EDTA, 125 mM DTT, 5% SDS, 2.5% bromophenol blue and 7.5% β-mercaptoethanol in water) and detached on ice using a cell scraper (BD Falcon) at 24, 48 and 72 h after transfection. The lysed cells were subjected to sonication for 10 s and centrifugation. After heating at 100°C for 5 min, the cell extracts were loaded into 10% SDS-PAGE and then transferred to PVDF membranes (EMD Millipore) using a Mini-protean Tetra (Bio-Rad). After 1 h blocking in 5% (w/v) BSA (Sigma-Aldrich) in TBST (50 mM Tris, 150 mM NaCl and 0.1% Tween 20), membranes were incubated with 1 μg/μl mouse anti-HA primary antibody (Covance, MMS-101P) overnight at 4°C, followed by incubation with a secondary HRP-conjugated antibody for 1 h at room temperature (RT). Proteins were visualized with a GelDoc system (Bio-Rad) by adding the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific).
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