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HIV-1 Fusion Assay Tailored for Measuring simultaneously HIV entry Into CD4 T cells, Monocytes, pDCs, from PBMCs

Authors: Nandhini Raman and Marielle Cavrois, updated date: , view: 19, Q&A: 0
Tags: PBMCs, HIV and Fusion Assay

Procedure

HIV-1 Fusion Assay Tailored for Measuring simultaneously HIV entry

Into CD4 T cells, Monocytes, pDCs, from PBMCs

 

Principle and Notes:

  • Ficoll cells

  • HIV Fusion Assay to measure HIV entry

  • Staining with antibody panel tailored for gating on possible HIV target cells (Into CD4 T cells, Monocytes, pDCs…)

 

Materials and Reagents

  • Reagent for Fusion Assay can be https://bio-protocol.org/e1212

  • Antibodies (See Table)

  • BD Bright Violet buffer

  • Ficoll-Paque

  • Leukoreduction Chamber from Trima Apheresis Collection (blood center of the pacific) http://www.bloodcenters.org/hospitals-research/products-services/

 

Equipment

  • FACS Aria with 5 lasers

  • Centrifuges


Procedure

A-Ficoll

  1. Wipe exterior of blood pack with EtOH

  2. Prepare scissors (clean with ethanol)

  3. Prepare a Falcon 50 ml tube

  4. Transfer blood from TRIMA into the falcon tubes by cutting first the bottom tube then the top let the blood drip

  5. Add to PBS to obtain 40 ml

  6. Dilvide the 40 ml into 2 flacon 50 (ie transfer out 20 ml in a fresh tibe)

  7. Add PBS to 35 ml in both tubes

  8. 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.

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

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

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

  12. Add 20 ml PBS.

  13. Pellet, wash twice

  14. Lead to about 500 million cells

 

B-Freeze the single cell suspension:

  1. Freeze PBMCs 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 for CX (1vial), 1ml for Cyto (2 vials), 1ml for EMB (2 vials), 1ml PBMCs (2 vials))

  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)

 

C- Fusion assay (see https://bio-protocol.org/e1212 for details about viral prep and assay solution composition)

  1. Thaw and count cells

  2. Transfer 4 million cells

  3. Infect: we use the primary C7, C17 and C24 viruses (500 ng p24Gag per well for 1.5h

  4. BlaM part (don’t forget the CTL for compensation) incubate overnight

 

D- Immunostaining

  1. Blocking non Specific binding

  2. Pellet the cells

  3. Dilute 5 µl of Fc block reagent into 100 µl of PBS-2% (per biopsies sample)

  4. Resuspend the pelleted cells in 100 µl of Fc block solution and transfer to a 96well plate with V bottom

  5. Incubate at room temp for 15 minutes at least

  6. Prepare the master mix staining panel by diluting the following antiB (100µl per samples is needed). Use eppendort tube and turn off light in BSC. Example for 10 samples

  7. In a tube an Eppendorf, prepare 100µl of staining solution (50µl of the BD buffer +50µl FACS buffer). Add Antibodies

  8.         1 ul-CD3-BUV737 T cells

            1 ul CD4-BUV395 CD4 (SK3)

            0.5 ul CD14_BV650 Mono/Macrophages (M1)

            ul HLA-DR-PerCP5.5 HLA-DR (APCs + activation marker)

            2 ul CD1c-PE

            1 ul Live dead Green 1/100*

            1ul CD303-PE-Cy7

            1 ul Lin APC (of each CD56, CD20 and CD19)

            1 ul CD141-APC-Cy7

            1 ul CD45ROECD 

    *Note that this is high dilution because we use FITC channel to detect the dead and that this channel has large contribution from CCF2 (this avoid to take an independent channel)

  9. Preparation of Comp Beads

    1. Add 1 drop of compbeads blue and 1 drop of the white cap (Make sure that wll antibodies are mouse against human)

    2. Pellet the beads 5 min at 1500 rpm

    3. Add antibody at 1/100 dilution, except for CD207_PE (use a 1/200 dilution to avoid saturating the signal) Note that for compensation the dilution of antibody does not have to match the dilution used in the staining mix

  10. Prepare the live dead CTLs:

    1. Take any cells (~0.1-0.5 million)

    2. Spin to concentrate them and transfer into 1 well of the 96 well plate

    3. Staining of cells with live dead control

  11. Stain for 30 min in dark at Room temp,

  12. Pellet add 150 µl of FACS Buffer and spin again.

  13. Fix in PFA and keep for at least 3 days (better discrimination of fusion)

  14. Fiter through FACS blue cap tubes just before FACS the sample

  15. FACS without fixation (Fig 2 gating strategy)


Data analysis

 Screen Shot 2018-06-25 at 8.11.32 AM.png



Used in:

unpublished;

 

 


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