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HIV-1 Fusion Assay Adapted for Measuring HIV entry into Cells from Female Reproductive Tract (ie endometrium and cervix)

Authors: Nandhini Raman and Marielle Cavrois, updated date: , view: 15, Q&A: 0
Tags: HIV and Fusion Assay

Procedure

HIV-1 Fusion Assay Adapted for Measuring HIV entry

into Cells from Female Reproductive Tract (ie endometrium and cervix)

 

Principle and Notes:

Prepare a single cell suspension of endometrium biopsies or ectocervix

Optional: Immunostain an aliquot to assess Red blood cells contamination, survey cells present and Trucount to get an idea of the number of cells collected

Freeze cells (if needed for work on cohorts)

HIV Fusion Assay to measure HIV entry

Staining with antibody panel tailored for gating on possible HIV target cells (CD4 T cells, Macrophages, DCs and Langerhans cells)

 

Materials and Reagents

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

Antibodies (See Table)

Blue Mr Freeze container

Digestion buffer

Trucount

BD Bright Violet buffer

 

Equipment

FACS Aria with 5 lasers

Centrifuges

 

Procedure

A-Preparation of single cell suspension from Endometrium (EMB) and endocervical (CX) biopsies

  1. Collect sample and place the biopsies in a 5ml Falcon RPMI 10% FBS Penn Strep

  2. Note: other antibiotics might be needed if the transport of samples in longer than 2h

  3. Note: Cut the biopsies if bigger than 2mm, especially the CX

  4. Spin down the biopsies 1500 rpm for 5 min

  5. Resuspend biopsies in 1ml of PBS without Ca&Mg for CX and 2 ml for EMB

  6. Add 1 (CX) or 2 ml (EMB) of the 2X solution with Collagenase and hyaluronidase

  7. Digest up to 1h30 at 37˚C

    Note: after 30 min, pipet up and down with a 5ml pipet).

    Note: Stop the digestion when samples looks digested (ie no more visible tissue for EMB and for CX the tissue becomes white)

  8. Filter single cell suspension filter through the 40 µm (use the mesh that fit on falcon 50 ml)

  9. Spin down cells

  10. Resuspend in 10 ml of PBS 2%FBS

  11. Take aliquot (usually 10% of total amount) for the immunostaining to determine blood contamination

 

B-Freeze the single cell suspension:

  1. Pellet the 9 ml remaining from the single cell suspension of EMB or CX

  2. Carefµlly remove the supernatant and resuspend in Freezing Media (90 FBS +10% DMSO)

  3. 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))

  4. 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-Immunostaining with “fresh Panel” for RBC count and get an idea of cell composition and cell number:

  1. Blocking non Specific binding

  2. Optional Transfer the cells into a TRucount tubes (make sure to write down the lot number as each lot has different count of beads

  3. Pellet the cells and beads

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

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

  6. Incubate at room temp for 15 minutes at least

  7. Prepare the master mix staining panel by diluting the following antiB (100µl per samples is needed ~200µl). Use eppendort tube and turn off light in BSC.

  8. In a tube an Eppendorf, comnbine 100 µl of BD brilliant violet buffer and 100 µl of PBS2%FBS

  9. Add antibodies, mix and keep in dark

    1. 4µl-CD326 (EpCAM) BV605 epithelial cells (1/50)

    2. 4µl-CD140b (PDGFPR) PerCP5.5 Stromal fibroblast (1/50).

    3. 4µl-CD235a-FITC red blood cells(1/50).

    4. 2µl-CD45-APC leucocytes (1/100)

    5. 2µl-CD3-BUV737 T cells (1/100)

    6. 2µl-CD4-BUV395 CD4 (1/100)

    7. 1µl-CD14-QDot655 Monocytes/macrophages (1/200)

    8. 4µl-CD1a: A700 Langerhans and mucosal DCs (1/50)

    9. 4µl-langPE Langerhans cells (1/50)

    10. 2µl of 1/10 dilution of LD Violet Dead cells (1/1000)

  10. 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

  11. Prepare the live dead CTLs:

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

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

  14. Staining of cells, comp beads and live dead control

  15. After 15 minutes of Fc block, Spin cells from C-1, Spin the bead from C3, Spin the CTL from C4

  16. Add 100 µl of the staining mix (step C2) to the cells, 100µl of single stain to beads, 100 µl of Live dead cell stain to cells at 1/1000 dilution (of DMSO resuspended dry pellet from the Invitrogen vial

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

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

  19. FACS without fixation (Fig 1 gating strategy)

 

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

  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. Transfer appropriate volume of thawed cell suspension 400,000 cells of EMB and 100,000 cells for CX

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

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

 

E- Immunostaining

  1. Blocking non Specific binding with Fc

    1. Pellet the cells

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

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

    4. Incubate at room temp for 15 minutes at least

  2. 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

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

    2. Add antibodies

    3. 1µl-CD3-BUV737 T cells

    4. 1µl CD4-BUV395 CD4

    5. 0.5µl CD14_BV650 Mono/Macrophages (M1)

    6. 1µl of CD45_BV605 Immune cells

    7. 0.5 µl HLA-DR-PerCP5.5 HLA-DR (APCs + activation marker)

    8. 2µl CD207 lang_PE Langerhans cells

    9. 1µl CD45_RO_ECD Naïve/Memory

    10. 1µl CD163-PE-Cy7 macrophages (M2)

    11. 1µl CD19, CD20 and CD56 APC hematopoietic stem cells

    12. 2µl-CD1a: A700 Langerhans and mucosal DCs

    13. 1µl-CD69-APC7 Activation marker

    14. 1µl Green Live Dead dead cells

    15. 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)

  3. 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

  4. 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, comp beads and live dead control

  5. Fix in PFA 1.2% in FACS buffer and keep for at least 3 days (better discrimination of fusion)

  6. Filter through FACS blue cap tubes just before FACS the sample



Digestion buffer and media

Digestion buffer 

Collagenase Type I – Worthington 4196.  249 units/mg.    1 gram total.

HBSS with Mg++ and Ca++.                                                          156ml

Hyaluronidase (Sigma H2251) – 20ku/ml.                                3.12ml

Penstrep                                                                                             3ml

 

 

Data analysis

Screen Shot 2018-06-23 at 3.58.35 PM.png


Used in:

unpublished;

 


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