Viromer® RED

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For adherent cells, you can target up to 80% confluency at the time of analysis.	Always add Buffer RED to Viromer® RED, not vice versa!

Upstream tips
For adherent cells, you can target up to 80% confluency at the time of analysis.
Protocol tips
Always add Buffer RED to Viromer® RED, not vice versa!

Publication protocol

Cell infection, transduction, and transfection
DC were exposed to the indicated HIV strains at a concentration ranging from 100 to 500 ng/ml p24 for 3–4 h at 37°C, washed, and cultured in medium with IL-4 and GM-CSF. In the HIV plus antiretroviral therapy (ART) conditions, DC were loaded as before with HIV but in the presence of 5 μM azidothymidine (AZT) and 1.2 μM nevirapine (NVP). AZT, NVP, and cytokines were maintained in the culture medium and renewed every second day.

At the indicated time points, DC infection was monitored using anti-Gag Ab (KC57-FITC or -PE, from Beckman Coulter) and flow cytometry. Cell transductions were performed as described (57) in the presence of SIV-Vpx (provided by A. Cimarelli, Centre International de Recherche en Infectiologie, Lyon, France) to enhance the transduction efficiency. Transduction efficiency was monitored by flow cytometry using GFP expression or Strawberry for pGIPZ shRNA lentiviral vectors or the expression of Atg4BC74A, respectively (55). HeLa-CIITA cells stably expressing pGIPZ shRNA-LC3 or pAtg4BC74A were generated by transduction and subsequent selection using puromycin (3 μg/ml). We obtained homogeneous populations of HeLa-CIITA cells with 100% GFP+ or Strawberry+ cells following transduction with shRNA-LC3 or Atg4BC74A, respectively. HeLa-CIITA cells were transfected with pHIVYu2b or pTRIP-CMV-Gag plasmids using calcium phosphate (58) or Viromer (Lipocalyx).

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Manufacturer protocol

Download the product protocol from Lipocalyx GmbH for Viromer® RED below.

Download manufacturer protocol

Videos

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