Author: Paquin, Ashley; Onabajo, Olusegun O.; Tang, Wei; Prokunina-Olsson, Ludmila
Title: Comparative Functional Analysis of 12 Mammalian IFN-?4 Orthologs Document date: 2016_1_1
ID: sqxrwgif_21
Snippet: For initial screening, transfections were done for 48 h in 6-well plates with 350,000 cells/well. Constructs, which showed positive signals for MAB-IFN-l4, were further evaluated by transfections in 12-well plates with 200,000 cells/well, in triplicates. Cells were harvested, fixed for 30 min at room temperature with 100 mL of cytofix (BD Biosciences), and stained overnight in cytoperm (BD Biosciences) with 0.5 mg/mL of a-Halo, 1 mg/mL RAB-IFN-l4.....
Document: For initial screening, transfections were done for 48 h in 6-well plates with 350,000 cells/well. Constructs, which showed positive signals for MAB-IFN-l4, were further evaluated by transfections in 12-well plates with 200,000 cells/well, in triplicates. Cells were harvested, fixed for 30 min at room temperature with 100 mL of cytofix (BD Biosciences), and stained overnight in cytoperm (BD Biosciences) with 0.5 mg/mL of a-Halo, 1 mg/mL RAB-IFN-l4, or 2 mg/mL of MAB-IFN-l4. Secondary antibodies were donkey anti-mouse and donkey anti-rabbit Alexa Fluor 488 (1 mg/mL; Life Technologies). Cells were analyzed using multiparametric flow cytometry on a FACS Aria III (BD Biosciences) and FlowJo.10 software (Tree Star).
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