Tales from the lab – HIV-Flow

In this most terrifying time of year, 

We’ve chosen to open and peer,

Into our well-guarded tomes of fear.


Between the recipe for washing solution blend, 

And the concoction for blocking, we recommend, 

Here’s the procedure, on which you can depend, 

For your double-labeled HIV samples to send.


Follow the formula, don’t forget,

With your pipette, be precise and set,

To isolate, it’s your best bet,

The double-p24+ in your HIV duet

Based on Pardons et al. (2019, PLoS Pathog) Single-cell characterization and quantification of translation-competent viral reservoirs in treated and untreated HIV infection

“The detection of p24+ cells by flow cytometry in clinical samples is technically challenging, since antibodies specific for HIV proteins are notorious for their limited specificity [Gadol, Crutcher & Busch, Cytometry. 1994; Kux et al. J Immunol Methods. 1996; Cameron et al. Cytometry. 1998].”



(Pardons et al. PLoS Pathog 2019; figure 2C)

HIV-Flow is a simple and effective method for quantifying HIV reservoirs competent for viral translation, by detecting cells expressing viral capsid protein (p24) by double labeling with anti-p24-APC (Medimabs cat.MM-0289-APC) and anti-p24 KC57-PE (Beckman Coulter cat.6604667). To perform this intracellular staining, the FoxP3 Transcription Factor Staining Buffer Set (eBioscience cat.00-5523-00) is used following the manufacturer’s instructions. This flow cytometry approach also enables these infected cells to be phenotyped.

(Pardons et al. PLoS Pathog 2019; figure 1C)


It was determined that the median number of p24+ cells per million CD4+ T cells (after PMA/Ionomycin stimulation) was found to be 4.3 in samples from ART participants, and ~100 in samples from untreated participants.


HIV-Flow Protocol (detailed protocol available online)

Day prior to staining

  • Thaw cryopreserved PBMCs (between 10-50 x 10^6 cells), whether from ART-treated or untreated participant samples.
  • Perform CD4+ T cell negative selection
  • Resuspend CD4+ T cells at a concentration of 2 x 10^6 cells/mL in RPMI + 10% FBS + Pen/Strep + ARV (to prevent new infection)
  • Perform stimulation:
    • For ART-treated samples: add PMA 162nM and ionomycin 1µg/mL, and incubate at 37ºc for 24 hours.
    • For untreated samples: add PMA 25nM and ionomycin 1µg/mL, and incubate at 37ºc for 18 hours. Untreated samples can also be staining without stimulation, but with an overnight resting.

**If using HIV Flow for immunophenotyping, add Brefeldin A (BFA) to prevent to the culture medium (dilution 2,000X) 1h prior to stimulation**


Day of staining

  • After prior stimulation time, collect cells in FACS tubes (maximum 10×10^6 cells per tube; 5 ml).
  • Wash by centrifugation and remove the supernatant by pipetting, carefully avoiding the cell pellet.
  • Perform Aqua Live/Dead staining in PBS (final volume of 200 μl per FACS tube) for 20-30 min at 4ºC, protected from light.
  • Add 800 μl PBS with 4% Human Serum (PBS/HS 4%) and wash by centrifugation.
  • Perform extracellular staining in PBS/HS 4% (final volume of 100 μl per FACS tube) for 30 min at 4ºC.
  • Add 1800 μl PBS/HS 4% and wash by centrifugation.
  • Perform Fix and Perm step by adding 100 μl of FoxP3 FixPerm Buffer for 45 min at 4ºC.
  • Add 1800 μl FoxP3 PermBuffer and wash by centrifugation.
  • Perform intracellular staining in FoxP3 PermBuffer (final volume of 100 μl per FACS tube) for 45 min at room temperature.

Prepare dilution of both anti-p24 antibodies in FoxP3 PermBuffer, without vortexing the antibodies or the mixes:

  • Dilution I (anti-p24 KC57-PE): 10-fold dilution (1 μl antibody + 9 μl FoxP3 Buffer)
  • Dilution II (anti-p24 28B7-APC): 10-fold dilution (1 μl antibody + 9 μl FoxP3 Buffer)
  • Dilution III: add 1 μl of each anti-p24 dilution per 100 μl (1 μl Dilution I + 1 μl Dilution II + 98 μl of FoxP3 PermBuffer)

**Please note that the anti-p24 antibodies need to be titrated upon reception of each new batch.**

  • Add 1800 μl FoxP3 PermBuffer and wash by centrifugation.
  • Resuspend in 100-200 μl PBS for Flow Cytometry analysis.


HIV Flow gating strategy

(Pardons et al. PLoS Pathog 2019; figure S13)


SSC-A + FSC-A lymphocyte gating → Single cells → Vivid- CD3+ cells → CD8- cells (include both CD4+ and CD4- since stimulation promotes CD4 downregulation) → double positive p24 (28B7+ and KC57+)

We wish you thrilling spells to cast, 

The tech team stands strong, from first to last. 

I hope the experiment was shadowy and vast, 

In our Tales from the lab, where terror is amassed!

Written by
MédiMabs’ Team
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