Studies in a Murine Model Confirm the Safety of Griffithsin and Advocate Its Further Development as a Microbicide Targeting HIV-1 and Other Enveloped Viruses

Kouokam, Joseph Calvin; Lasnik, Amanda B.; Palmer, Kenneth E.

To assess the expression of cellular surface activation markers, flow cytometry was performed after dual fluorescent staining with anti-mouse antibodies purchased from BD Biosciences TM (San Diego, CA, USA). Briefly, cultures were transferred from plates to 5 mL round-bottom tubes, and washed with PBS containing 5% inactivated FBS. Then, cells were blocked with purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block TM ; BD, San Jose, CA, USA) for 10 min followed by incubation in the dark with fluorescein isothiocyanate (FITC)-conjugated rat anti-CD4 mAb in combination with phycoerythrin (PE)-conjugated rat anti-CD25 or hamster anti-CD69 mAb for 30 min on ice. Finally, cells were washed and analyzed with a FACSCalibur (BD, San Jose, CA, USA). All flow cytometry data were acquired and analyzed using CellQuest Pro from BD, counting 10,000 events per sample. ConA (0.37 µM) and PHA (10 µg/mL) were used as positive controls [12, 18] , and PBS as the negative control.