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)
Prepare MACS buffer= PBS 2% FBS 2 mM EDTA
Pellet cells at 300g for 10 min in a 17 ml Falcon tube
Remove the sup completely
Add 80 ul of buffer per 107 cells (example 50 *80ul =4,000ul)
Add 20 ul of CD19 microbeads (example 20 *80ul =1,000ul)
Mix well
Incubate in refrigerator for 15 min
Wash cells by adding 4 ml of buffer, pellet
Resuspend in 4 ml of MACS buffer
Filter 40 um mesh
Prep column: Place column on the magnetic field LS column, rinse with 3ml of MACS buffer
Change collection tube to a 15 ml sterile falcon
Magnetic separation: Apply the 5ml cells by steps
Collect the flow through as the CD19+ cells will remain on the column
Wash 3 times the column with 3ml of buffer and continue to collect
Pellet and count (example yield 1,56 x 108 CD19 depleted HLACs cells)
Fusion Assay
Aliquot 50M cells per one 15 ml Falcon tubes and keep some leftover for the non infected control
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)
Pellet cells, wash with 10 ml of CO2 independent media, spin and resuspend in 1ml of CCF2 loading solution
Incubate 1 h at Room Temperature
Spin dowm cells and resuspend in 4 ml of development media
Incubate 6 hours
Pellet and Stain with the in vitrogen LIVE/DEADred dye for 15 min at the concentration recommended by manufacturer
Pellet cells, resuspend in ~4ml Filter through 40 um strainer
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)