For the quantification of MLV titers by coculture, round, 12-mm-diameter BioCoat coverslips (Corning; Fisher Scientific, Merelbeke, Belgium) were first added to a 24-well plate (Nunc; Thermo Scientific, Asse, Belgium). Next, approximately 1.5 × 105 NIH 3T3 cells were seeded into each well. After 24 h, NIH 3T3 cells were cocultured with 104 cells from the spleen or thymus for 24 h. Next, NIH 3T3 cells were rinsed with PBS and fixed with 4% paraformaldehyde. For staining, a primary antibody against the MLV protein p12 was obtained from hybridoma alpha CA cells (ATCC CRL-1890). The primary antibody was incubated for 1 h. Goat anti-mouse biotin (Dako Denmark, Glostrup, Denmark) was used as a secondary antibody, and samples were incubated for 20 min, followed by an incubation step with streptavidin-horseradish peroxidase (HRP) (Dako Denmark) for 30 min. Next, 3,3’-diaminobenzidine (DAB) staining was done. Coverslips were mounted using Mowiol 4-88 (Calbiochem, Merck). Infected cells were counted with a Leica DMR microscope (×40 magnification).
Hybridoma alpha ca cells
Manufactured by American Type Culture Collection
Hybridoma alpha CA cells are a type of immortalized cell line derived from the fusion of a mouse myeloma cell and a mouse B lymphocyte. These cells are used for the production of monoclonal antibodies and are a key tool in immunological research and diagnostics.
Automatically generated - may contain errors
3 protocols using hybridoma alpha ca cells
1
Quantifying MLV Titers by Coculture
2
Quantifying MLV Titers by Coculture
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For the quantification of MLV titers by coculture, round, 12-mm-diameter BioCoat coverslips (Corning; Fisher Scientific, Merelbeke, Belgium) were first added to a 24-well plate (Nunc; Thermo Scientific, Asse, Belgium). Next, approximately 1.5 × 105 NIH 3T3 cells were seeded into each well. After 24 h, NIH 3T3 cells were cocultured with 104 cells from the spleen or thymus for 24 h. Next, NIH 3T3 cells were rinsed with PBS and fixed with 4% paraformaldehyde. For staining, a primary antibody against the MLV protein p12 was obtained from hybridoma alpha CA cells (ATCC CRL-1890). The primary antibody was incubated for 1 h. Goat anti-mouse biotin (Dako Denmark, Glostrup, Denmark) was used as a secondary antibody, and samples were incubated for 20 min, followed by an incubation step with streptavidin-horseradish peroxidase (HRP) (Dako Denmark) for 30 min. Next, 3,3’-diaminobenzidine (DAB) staining was done. Coverslips were mounted using Mowiol 4-88 (Calbiochem, Merck). Infected cells were counted with a Leica DMR microscope (×40 magnification).
3
Quantifying Retroviral Infection in Co-Cultures
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First, rounded BioCoat TM coverslips 12 mm in diameter (Corning, Fisher Scientific, Merelbeke, Belgium) were added to a 24-well plate (Nunc, Thermo Scientific, Asse, Belgium). Then, approximately 1.5•10 5 NIH/3T3 cells were seeded in each well. After 24 hours, NIH/3T3 cells were co-cultured with 10 4 cells from spleen or thymus for 24 hours.
Then, NIH/3T3 were rinsed with PBS and they were fixed with 4% paraformaldehyde. For staining, a primary antibody against MLV protein p12 was obtained from hybridoma alpha CA cells (ATCC CRL-1890). Primary antibody was incubated for 1 hour. Goat antimouse biotin (Dako Denmark, Glostrup, Denmark) was used as a secondary antibody and samples were incubated for 20 min, followed by an incubation with streptavidin-HRP (Dako Denmark) for 30 min. Then, a DAB staining was done. Coverslips were mounted using Mowiol®4-88 (Calbiochem, Merck). Infected cells were counted with a Leica DMR microscope (40X magnification).
Then, NIH/3T3 were rinsed with PBS and they were fixed with 4% paraformaldehyde. For staining, a primary antibody against MLV protein p12 was obtained from hybridoma alpha CA cells (ATCC CRL-1890). Primary antibody was incubated for 1 hour. Goat antimouse biotin (Dako Denmark, Glostrup, Denmark) was used as a secondary antibody and samples were incubated for 20 min, followed by an incubation with streptavidin-HRP (Dako Denmark) for 30 min. Then, a DAB staining was done. Coverslips were mounted using Mowiol®4-88 (Calbiochem, Merck). Infected cells were counted with a Leica DMR microscope (40X magnification).
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