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

1. Prepare single cell suspension

2. Pace a 0.7 um strainer in a petri dish containing Tonsil media and add 10 ml tonsil media

3. Aspirate the media from the tube in which the tonsils biopsies have been sent

4. Cut the tonsil in small piece about 2mm

5. Transfer the small pieces of tonsils into the 0.7 um strainer located it the petri dish

6. 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

7. Repeat until all the small piece are dispersed into the media

8. Transfer all the suspension into a 50 ml falcon. At the same time filter with a 40 um strainer

9. Bring the volume to 35 ml (either by adding media or by spin down and resuspending the cells 

B.Ficoll

1. Add to PBS to obtain 40 ml

2. Divide the 40 ml into 2 falcon 50 (ie transfer out 20 ml in a fresh tibe)

3. Add PBS to 35 ml in both tubes

4. 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.

5. Spin 30 minutes at 2000 rpm at RT, no brake

6. Remove ~10 ml of Supernatant (ie above the buffy)

7. Slowly pipet out the 5-10 ml of interface, with as little ficoll as possible,

8. add 20 ml PBS.

9. Pellet, wash twice

10. Lead to about 1 billion cells million cells

C. Freeze

1. Freeze HLACs that are not needed as they can be reuse

2. Pellet left over cells

3. Carefµly remove the supernatant and resuspend in Freezing Media (90 FBS +10% DMSO) for having 50 million cells per vial of 500ul

4. Transfer the cell solution into cryo vials (500µl per vial),

5. 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

1. Thaw and count cells

2. Thaw the vial in a water bath under completely melted

3. Transfer to a 15 ml falcon

4. Add RMI 10 ml

5. Count cells

6. Spin down and resuspend into 200ul

7. Aliquot the cells in the appropriate numbers of well.

8. 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

9. Infection

10. Pellet the cells by centrifugation and infect or not (not infected control) with HIV virions

11. Add virus at between 100 and 500 ng p24

12. Note: OK to use HIV containing BlaM-Vpr if doing the fusion assay in paralle

13. Incubate for 1h at 37 degree

14. Wash

15. 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)

16. Incubate for at least 3 days (can do more but will be several rounds of infection)

17. Staining (find HLAC experiment with intracellular p24)

18. At appropriate time, pellet cells

19. Staining first for 15 min with violet LIVE/Dead diluted at 1/500 in FACS buffer

20. Wash and Fix in PFA 1.2% (diluted in FACS buffer) for 1h at Room Temp or O/N at 4˚C.  

21. After fixing, pellet and wash once in PBS containing 2%FBS

22. Fc Block 5ul in 95ul for 15 min at room temp (no wash)

23. 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%

1. 1u of KC57Gag PE

2. 1ul-CD3-BUV737 T cells

3. 1ul CD4-BUV395 CD4 SK3 clone

4. 0.5ul CD14_BV650 Mono/Macrophages (FYI: there are no macrophage/monocyte in HLAC)

5. 1ul CD19-PerCP5.5

6. 1ul CD45PE-Cy7

7. 1ul CD56-APC

8. 1ulCD8ECD

24. Also stain comp beads. Use another PE antibody for PE if using the mousse BD comp beads because it is a rat anti body

25. 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