Virus titers were quantified via plaque assay. First, serial dilutions of virus were absorbed to confluent Vero cells for 1 h in a small amount of serum free DMEM. Virus was then removed from cells and an agarose overlay (equal volumes of 2× DMEM and 1.8% melted agarose (Invitrogen Corporation)) was added. After 2 d, an additional agarose overlay containing 0.015% neutral red (MP Biomedicals, LLC, Solon, OH) was added to cells. Approximately 24 h later, clear plaques were counted and virus titers were calculated in particle forming units/ml (pfu/ml).
Agarose
Manufactured by MP Biomedicals
Sourced in United States
Agarose is a purified polysaccharide derived from marine red algae. It is a gel-forming agent commonly used as a matrix in various laboratory applications, particularly in electrophoresis techniques for the separation and analysis of biomolecules such as DNA, RNA, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Agarose, by Thermo Fisher Scientific (1 mentions)
Neutral red, by MP Biomedicals (1 mentions)
Neutral red, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Collagen type 1, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Ficoll hypaque, by Merck Group (1 mentions)
Diazepam, by Merck Group (1 mentions)
Alexa flour 488 goat antimouse igg h l 2, by Thermo Fisher Scientific (1 mentions)
Omniscript rt kit, by Qiagen (1 mentions)
3 protocols using agarose
1
Virus Titer Quantification via Plaque Assay
2
MERS-CoV Plaque Assay for Titer Determination
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Infectious MERS-CoV titers were determined by plaque assays as described previously (19). Briefly, Vero cells were seeded in 6-well plates at 5 × 105 cells/well and cultured overnight at 37 °C in a 5% CO2 incubator. Cells were inoculated with ten-fold serially diluted cell culture supernatants for 1 h at 37 °C. After adsorption, cells were washed with PBS and overlaid with DMEM containing 0.5% agarose (MP Biomedicals, Solon, OH, USA) and 2% FBS. After three days of incubation, plaques were visualized by staining with 50 μg/mL neutral red (Sigma-Aldrich).
3
Immunophenotypic Characterization of Stem Cells
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The Collagen Type-I, diazepam, Ficoll-Hypaque, pentylenetetrazole (PTZ), and trypan blue were purchased from Sigma Chemicals (USA). The cell culture reagents were purchased from Invitrogen Corp. (CA, USA). For cell washing, physiological buffer i.e., 1x phosphate saline buffer (PBS) was used.
For immunostaining, monoclonal antibodies to rat H-CAM; CD44H (BD Pharmingen Company, Franklin Lakes, NJ, USA), and rat CD90 (Thy 1.1) (Cedarlane Laboratories Ltd. Canada) were used. Alexa flour® 488 goat anti-mouse IgG (H + L)*2 (Invitrogen Corporation Carlsbad, CA, USA) was used as secondary antibody. For RT-PCR analysis of CD markers, RNeasy Mini Kit (Qiagen Inc. Valencia, CA, USA), Superscriptase III First-Strand synthesis system for RT-PCR (Invitrogen Corporation Carlsbad, CA, USA) and Omniscript RT kit (Qiagen Inc. Valencia, CA) were used. For Agarose gel electrophoresis; Agarose, was purchased from MP Biomedicals. Inc, France and DNA ladder; Gene Ruler™, 100bp DNA ladder was purchased from Fermentas. Inc. (Glen Burnie, MD, USA). The GAPDH was used as an internal standard (house keeping gene). The primer sequence of the mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene, CD44 and CD90 are shown in table 1.
For immunostaining, monoclonal antibodies to rat H-CAM; CD44H (BD Pharmingen Company, Franklin Lakes, NJ, USA), and rat CD90 (Thy 1.1) (Cedarlane Laboratories Ltd. Canada) were used. Alexa flour® 488 goat anti-mouse IgG (H + L)*2 (Invitrogen Corporation Carlsbad, CA, USA) was used as secondary antibody. For RT-PCR analysis of CD markers, RNeasy Mini Kit (Qiagen Inc. Valencia, CA, USA), Superscriptase III First-Strand synthesis system for RT-PCR (Invitrogen Corporation Carlsbad, CA, USA) and Omniscript RT kit (Qiagen Inc. Valencia, CA) were used. For Agarose gel electrophoresis; Agarose, was purchased from MP Biomedicals. Inc, France and DNA ladder; Gene Ruler™, 100bp DNA ladder was purchased from Fermentas. Inc. (Glen Burnie, MD, USA). The GAPDH was used as an internal standard (house keeping gene). The primer sequence of the mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene, CD44 and CD90 are shown in table 1.
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