Measurement of HIV Productive Infection of HLACs
by FACS
Principle and Notes
Human lymphoid aggregate culture (HLACs) are great to study HIV as there is no need for activation of the cells to render the cells permissive (as for blood)
Down regulation of CD4 by HIV accessory protein lead to the absence of CD4 on the productively infected cells.
Procedure: Cell prep and infection and detection
Applicable to activated T cells in Blood and can be combine with fusion assay if infection is done with more cells and an aliquot is kept to do the fusion assay
Procedure
A.Prepare HLACs culture
Prepare single cell suspension
Pace a 0.7 um strainer in a petri dish containing Tonsil media and add 10 ml tonsil media
Aspirate the media from the tube in which the tonsils biopsies have been sent
Cut the tonsil in small piece about 2mm
Transfer the small pieces of tonsils into the 0.7 um strainer located it the petri dish
With the plunger of the 10 ml syringe, push the tonsil against the side of the filter to disperse the tonsil into a single cell suspension in the media outside the filter (do this until only the extracellular matrix, connective tissue and fat remain
Repeat until all the small piece are dispersed into the media
Transfer all the suspension into a 50 ml falcon. At the same time filter with a 40 um strainer
Bring the volume to 35 ml (either by adding media or by spin down and resuspending the cells
B.Ficoll
Add to PBS to obtain 40 ml
Divide the 40 ml into 2 falcon 50 (ie transfer out 20 ml in a fresh tibe)
Add PBS to 35 ml in both tubes
Add 12.5 ml Ficoll to two new 50 ml falcon tubes and Layer blood on top, being careful to not let any stick to the sides.
Spin 30 minutes at 2000 rpm at RT, no brake
Remove ~10 ml of Supernatant (ie above the buffy)
Slowly pipet out the 5-10 ml of interface, with as little ficoll as possible,
add 20 ml PBS.
Pellet, wash twice
Lead to about 1 billion cells million cells
C. Freeze
Freeze HLACs that are not needed as they can be reuse
Pellet left over cells
Carefµly remove the supernatant and resuspend in Freezing Media (90 FBS +10% DMSO) for having 50 million cells per vial of 500ul
Transfer the cell solution into cryo vials (500µl per vial),
Place the cryovials in the blue Mr Freeze container previously filled with appropriate level of isopropanol. Freeze at -80 over night and next day transfer to the Liquid nitrogen container (log the sample in the google excel file)
B. Infection, and Staining for intracellular HIV Gag
Thaw and count cells
Thaw the vial in a water bath under completely melted
Transfer to a 15 ml falcon
Add RMI 10 ml
Count cells
Spin down and resuspend into 200ul
Aliquot the cells in the appropriate numbers of well.
Note: Max 4 million in one well for infection and for incubation need to take the wells in duplicate and combine at the end after staining
Infection
Pellet the cells by centrifugation and infect or not (not infected control) with HIV virions
Add virus at between 100 and 500 ng p24
Note: OK to use HIV containing BlaM-Vpr if doing the fusion assay in paralle
Incubate for 1h at 37 degree
Wash
Resuspend in 200 ul of Tonsil Media. Make sure to use only 2M cells per well, avoid the outside of the 96w plate (or add PBS on the outside wells)
Incubate for at least 3 days (can do more but will be several rounds of infection)
Staining (find HLAC experiment with intracellular p24)
At appropriate time, pellet cells
Staining first for 15 min with violet LIVE/Dead diluted at 1/500 in FACS buffer
Wash and Fix in PFA 1.2% (diluted in FACS buffer) for 1h at Room Temp or O/N at 4˚C.
After fixing, pellet and wash once in PBS containing 2%FBS
Fc Block 5ul in 95ul for 15 min at room temp (no wash)
Spin down cells and stain for 30 min to 1h at R temperature with 100ul of (50ul of the BD buffer +50ul FACS buffer) +10ul of Saponin 1%
1u of KC57Gag PE
1ul-CD3-BUV737 T cells
1ul CD4-BUV395 CD4 SK3 clone
0.5ul CD14_BV650 Mono/Macrophages (FYI: there are no macrophage/monocyte in HLAC)
1ul CD19-PerCP5.5
1ul CD45PE-Cy7
1ul CD56-APC
1ulCD8ECD
Also stain comp beads. Use another PE antibody for PE if using the mousse BD comp beads because it is a rat anti body
Fix and FACS
Data analysis

Figure: Gating strategy: Top 2 raws are infected with 2 TF founder HIV virus (MC code C7 and C37)
Explain that CD4 is down modulated by HIV accessory proteins (Nef and VPU) also by Env. So, the productive infected are CD4-
Recipes
Enter text here.
Tonsil Media, consisting of RPMI supplemented with 15% heat-inactivated fetal bovine serum (FBS), 100 g/ml gentamicin, 200 g/ml ampicillin, 1 mM sodium pyruvate, 1% non-essential amino acids (Mediatech), 1% Glutamax (Thermo Fisher), and 1% Fungizone (Invitrogen).
FACS buffer
Make saponin buffer at 1% stock in PBS