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Flow cytometry analysis of cell-associated Gag levels

JD Jérémy Dufloo
CP Cyril Planchais
SF Stéphane Frémont
VL Valérie Lorin
FG Florence Guivel-Benhassine
KS Karl Stefic
NC Nicoletta Casartelli
AE Arnaud Echard
PR Philippe Roingeard
HM Hugo Mouquet
OS Olivier Schwartz
TB Timothée Bruel
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Cells were washed and incubated with a PE-conjugated anti-CD4 antibody (clone VIT4; Miltenyi; dilution 1:200) for 30 min at 4 °C. Cells were fixed with 4% PFA for 10 min at room temperature. Cells were washed and stained with a FITC-conjugated anti-Gag antibody (clone KC57; Beckman-Coulter; diluted 1:500 in PBS/BSA 1%/Saponin 0.05%) for 30 min at room temperature. Cells were washed and resuspended in PBS. Data were acquired on an Attune NxT flow cytometer (Life Technologies)using the Attune NxT Software (Life Technologies). Data were analyzed using the FlowJo software (v10.7; BD). The Median Fluorescence Intensity (MFI) of Gag in infected (CD4−Gag+) cells was calculated and normalized to the condition without antibody.

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