After euthanasia, spleens were removed from mice and transferred to a 70-μm cell strainer placed on top of a 50 mL tube. The tissue was pushed through the strainer using a 10 mL syringe plunger and washed with 10 mL ice-cold DMEM (Corning®) to make a single cell suspension. Cells were collected by centrifuge (400 g, 5 min), and red blood cells were removed by Red Blood Cell Lysis Buffer (Biolegend). Splenic cells were then resuspended in DMEM, counted on TC20™ Automated Cell Counter (Bio-Rad Laboratories) and kept on ice for flow cytometry staining.
Dulbecco modified eagle medium (dmem)
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
DMEM (Dulbecco's Modified Eagle's Medium) is a commonly used cell culture medium developed by Harry Eagle. It is a nutrient-rich basal medium designed to support the growth and maintenance of a wide variety of cell types in vitro. DMEM contains essential amino acids, vitamins, salts, and glucose to provide the necessary components for cell proliferation and survival.
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Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Ln229, by American Type Culture Collection (2 mentions)
U87mg, by American Type Culture Collection (2 mentions)
Dnase, by Bio-Rad (2 mentions)
Apc conjugated isotype controls, by BioLegend (2 mentions)
Fc blocking antibody, by BioLegend (2 mentions)
Apc conjugated anti f4 80, by BioLegend (2 mentions)
Anti cd140b, by BioLegend (2 mentions)
Collagenase 2, by Merck Group (2 mentions)
Facsaria 2, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Baby rabbit complement, by Cedarlane (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Trypsin, by Merck Group (2 mentions)
Red blood cell lysis buffer, by BioLegend (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Tc20 automated cell counter, by Bio-Rad (1 mentions)
12 protocols using dulbecco modified eagle medium (dmem)
1
Isolation and Preparation of Splenic Cells
2
Culturing Human Glioma Cell Lines
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Human glioma cell lines (U87MG, U251, and LN229) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Bio-Rad, Hercules, CA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher, Waltham, MA, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C under a humidified 5% CO2/95% air atmosphere. Human microglia cells (Sciencell Research Laboratories, Carlsbad, CA, USA) were used as the control.
3
Cellular Imaging of Nanoparticle Uptake
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After 1-h exposure to NPs, the cells were stained with 20 nM PureBlu Hoechst 33342 in DMEM (BioRad, Hercules CA, USA) for 5 min at 37°C and washed thrice with PBS (2.5% FBS). Cells were then fixed with 4% paraformaldehyde/PBS (Sigma-Aldrich) for 30 min at 37 °C and washed. SEM images were obtained using a Hitachi S-3400N scanning electron microscope equipped with a Thermo Scientific UltraDry EDS detector. SEM images are provided with a description of the measurement conditions. Microscopic images were taken using an AxioObserverZ1 inverted fluorescence wide-field microscope (Carl Zeiss, Oberkochen, Germany) with an LD40×/0.4 Korr Ph2 objective. A BF Condenser (NA = 0.4) was used to measure bright field images, while the upconverted emission of NaGdF4:Yb3+,Er3+ was captured using a 975 nm laser diode (Spectra-Laser, Opole, Poland) excitation with a customized optical setup. Filter cube was composed of FF750-SDi02 dichroic and FF01-945 emission filter to cut the 976-nm excitation and transmit visible radiation. CCD AxioCam MRc5 (Carl Zeiss) and EMCCD Rolera EM-C2 (QImaging, Surrey, BC, Canada) as well as ZEN2011 (Carl Zeiss) software were used to document images.
4
Culturing Human Glioma Cell Lines
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Human glioma cell lines (U87MG, U251, and LN229) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Bio-Rad, Hercules, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were incubated in an atmosphere of 37°C with 5% CO2.
5
Isolation of Kidney Stromal Cells
6
Interferon-Alpha Modulation of HSV-1 Infection in HUVECs
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HUVECs were pretreated with or without 2-fold serial dilution of IFN-α starting from 1,000 to 1.8 U/mL for 24 h. HSV-1 at a multiplicity of infection (MOI) of 0.1–64 was added to the medium containing the cells using opti-MEM (Thermo Scientific) for 1 h at 37°C. Viral supernatant was then removed, and the cells were refreshed with complete medium. The medium was removed 48 h of post infection and cells were fixed with 10% formaldehyde solution for 20 min at room temperature. After fixation, cells were visualized with 0.4% crystal violet. The excessive dye was then removed by immersing the plate in PBS. Each treatment was performed in duplicate. For plaque formation assay to measure the MOI of HSV-1, different dilutions of supernatant from virus-infected cells were used to infect Vero cells in opti-MEM for 1 h, followed by overlaying 2% FBS in DMEM containing 1% agarose (Bio-Rad) to immobilize the virus. After 24 h, cells were fixed and visualized with crystal violet, and the plaques were enumerated.
7
Neuroinflammation Assay Protocol
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DMEM, heat inactivated FBS, Hank's balanced salt solution (HBSS) and PBS were purchased from Bio-Rad Laboratories, Inc. Penicillin/streptomycin sulfate, amphotericin B and L-glutamine were purchased from Mediatech, Inc. Cocaine hydrochloride (Ecgonine methyl ester benzoate; molecular weight, 339.8), trypan blue, sulfanilamide, LPS from Escherichia coli serotype 0111:B4, N-(1-naphthyl)-ethylenediamine (NED), phosphoric acid, and EDTA were supplied by Sigma-Aldrich; Merck KGaA. IFNγ protein was obtained from Novus Biologicals Ltd. All other routinely used agents were analytical grade.
8
SARS-CoV-2 Quantification Using Plaque Assay
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HcoV-229E was quantified with standard plaque assay protocols [19 (link)]. HEK-293 cells (ATCC, Cat. No. CRL-1573) were seeded at 2.0 × 105 cells per well in 6 well tissue culture treated plates (Fisher Science, Cat. No. 07–200-601) with 2 mL of DMEM (Fisher Scientific, Cat. No. 11–965-118) infused with 5% heat inactivated fetal bovine serum (Gemini Bioproducts, Cat. No. 100–500). Once the cells achieved 85% confluence, 200 μL of HcoV-229E stock was added to the media of the first well. The media was gently mixed and 200 μL of the infected media was transferred into the adjacent well. This was repeated to create serial dilutions throughout the plate. After 24 h of incubation, the virally infected media was removed from each well and replaced with 2 mL of DMEM infused with 5% heat inactivated FBS and 2% agarose (Bio-Rad, Cat. No. 1613101). The plate was incubated for an additional 5 days at 35 °C with 5% CO2 and plaques were counted to determine viral concentration in plaque forming units/mL (PFU/mL).
9
Electroporation-Mediated RNAi in O. viverrini Flukes
10
Isolation of Kidney Stromal Cells
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For tissue preparation, the obstructed kidney was minced and incubated with 2mg/mL collagenase II (Sigma), 1mL DMEM, and DNAse (BioRad 10ul/mL) in a shaker at 37C for 1 hour. The tissue was filtered (70 μm), centrifuged, and incubated in red blood cell lysis buffer (15.5mM NH4Cl, 1mM KHCO3, 10 μM EDTA) at 37C for 5 minutes, neutralized with PBS, centrifuged, and resuspended in PBS with 1%FBS. The tissue was passed through a 50 μM strainer, and tissue from COL-Cre mice (and controls) was passed through a 24 μM filter to remove the glomeruli. The FACSAria II from BD Biosciences in the Research Flow Cytometry Core Laboratory at the Nashville VA Medical Center was used for sorting of GFP+ cells.
For flow cytometry, samples were incubated with Fc blocking antibody (Biolegend) on ice, then incubated with APC-conjugated anti-F4/80, anti-CD140b (PDGFRβ), or the appropriate APC-conjugated isotype controls (Biolegend) for 30 minutes at room temperature. Cells were analyzed on a FACS Canto II cytometer with FACSDiVa v6.1 software for data acquisition and analysis at the Nashville VA Medical Center’s Flow Cytometry Laboratory.
For flow cytometry, samples were incubated with Fc blocking antibody (Biolegend) on ice, then incubated with APC-conjugated anti-F4/80, anti-CD140b (PDGFRβ), or the appropriate APC-conjugated isotype controls (Biolegend) for 30 minutes at room temperature. Cells were analyzed on a FACS Canto II cytometer with FACSDiVa v6.1 software for data acquisition and analysis at the Nashville VA Medical Center’s Flow Cytometry Laboratory.
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