RSV culture

AL Annefleur C. Langedijk
RL Robert Jan Lebbink
CN Christiana Naaktgeboren
AE Anouk Evers
MV Marco C. Viveen
AG Anne Greenough
TH Terho Heikkinen
RS Renato T. Stein
PR Peter Richmond
FM Federico Martinón-Torres
MN Marta Nunes
MH Mitsuaki Hosoya
CK Christian Keller
MB Monika Bauck
RC Robert Cohen
JP Jesse Papenburg
JP Jeffrey Pernica
MH Marije P. Hennus
HJ Hong Jin
DT David E. Tabor
AT Andrev Tovchigrechko
AR Alexey Ruzin
MA Michael E. Abram
DW Deidre Wilkins
JW Joanne G. Wildenbeest
LK Leyla Kragten-Tabatabaie
FC Frank E. J. Coenjaerts
ME Mark T. Esser
LB Louis J. Bont
ask Ask a question
Favorite

Frozen respiratory samples stored in UTM are thawed, combined 1:1 with DMEM (Dulbecco’s Minimal Essential Medium; Lonza) supplemented with 5% FBS and 100 μg/ml Normocin (InvivoGen), and subsequently filtered through a 0.45 μm filter. The filtrate is used to infect HEp-2 cells (60% confluent) in T25 flasks for 1 h at 33 °C and 5% CO2. The supernatant is replaced with fresh DMEM supplemented with 5% FBS and 100 μg/ml Normocin and placed back into the 33 °C humidified, 5% CO2 incubator. The viral culture is harvested upon reaching approximately 70% cytopathic effect (CPE) by centrifugation at 247×g for 10 min and combining the supernatant with 50% sucrose in dPBS (sterile filtered). The viruses are stored in 1 ml aliquots at − 80 °C.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A