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Sorting HLACs cells that supported fusion of HIV virions
Author: Marielle Cavrois, updated date: , view: 32, Q&A: 0
Tags: HIV, Fusion Assay and HLACs

Procedure

Sorting HLACs cells that fused HIV virions

 

Generation of HLACs from tonsil tissues (as described Cavrois et al. Cell Report 2017)

FACS buffer Generation of HLACs from tonsil tissues Human tonsils from the Cooperative Human Tissue Network (CHTN) were dissected into 4 mm3 pieces, passed through a 40-μm strainer, and cultured in 96-well U-bottomed polystyrene plates (106 cells/well) in 200 μl/well of Tonsil Media, consisting of RPMI supplemented with 15% heat-inactivated fetal bovine serum (FBS), 100 μg/ml gentamicin, 200 μg/ml ampicillin, 1 mM sodium pyruvate, 1% non-essential amino acids (Mediatech), 1% Glutamax (Thermo Fisher), and 1% Fungizone (Invitrogen). Following isolation, HLACs were immediately used for viral fusion or infection assays. Of note, HLAC cells were never stimulated with a mitogen at any point during in vitro culture.

Ficoll the HLACs cells count cells (should be ~2 to 5 x 108 cells, example is 5 x 108 cells)

Depletion of B cells with Miltenyi CD19 positive selection kit (To shorter the sorting time, CD19+ B cells are removed from the HLACs cell suspension). If using a R5 tropic HIV, CD45RA+ cells, which includes B-Cells and naïve T cells can be removed as they don’t support fusion of R5-HIV)

      1. Prepare MACS buffer= PBS 2% FBS 2 mM EDTA

      2. Pellet cells at 300g for 10 min in a 17 ml Falcon tube

      3. Remove the sup completely

      4. Add 80 ul of buffer per 107 cells (example 50 *80ul =4,000ul)

      5. Add 20 ul of CD19 microbeads (example 20 *80ul =1,000ul)

      6. Mix well

      7. Incubate in refrigerator for 15 min

      8. Wash cells by adding 4 ml of buffer, pellet

      9. Resuspend in 4 ml of MACS buffer

      10. Filter 40 um mesh

      11. Prep column: Place column on the magnetic field LS column, rinse with 3ml of MACS buffer

      12. Change collection tube to a 15 ml sterile falcon

      13. Magnetic separation: Apply the 5ml cells by steps

      14. Collect the flow through as the CD19+ cells will remain on the column

      15. Wash 3 times the column with 3ml of buffer and continue to collect 

      16. Pellet and count (example yield 1,56 x 10CD19 depleted HLACs cells)

Fusion Assay

      1. Aliquot 50M cells per one 15 ml Falcon tubes and keep some leftover for the non infected control

      2. Infection with 240ul of BlaM-Vpr containing virions for 2 h at 37˚C (see procedure described in bio-protocol https://bio-protocol.org/e1212)

      3. Pellet cells, wash with 10 ml of CO2 independent media, spin and resuspend in 1ml of CCF2 loading solution

      4. Incubate 1 h at Room Temperature

      5. Spin dowm cells and resuspend in 4 ml of development media

      6. Incubate 6 hours

      7. Pellet and Stain with the in vitrogen LIVE/DEADred dye for 15 min at the concentration recommended by manufacturer

      8. Pellet cells, resuspend in ~4ml Filter through 40 um strainer

      9. Sort under BSL3 biohazarodus containement

Gating strategy 

The strategy is given in the supplemental figure (Figure S2) of our paper (Cavrois et al. Mass Cytometric Analysis of HIV Entry, Replication and Remodeling in Tissue CD4+ T CellsCell Report 2017)


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