Standard Curve. == Acknowledgments == We would like to thank Haydenet al. for developing the FLAQ assay. == References ==. sorting (FACS) staining buffer (see Recipes) == Equipment == Eppendorf centrifuge Rotating mixer 96-well V-bottom plate (optional) (Thermo Fisher Scientific, catalog number: 12-565-216) Thermo Fisher lids for 96-well microplates (Thermo Fisher Scientific, catalog number: 14-245-53A) Plate sealers (VWR International, catalog number: 62402-921) Eppendorf tubes Multichannel pipetman (optional) Pipetman Tips (1 Aceclofenac ml, 200 l, and 10 l) Reagent reservoirs (optional) Tissue culture centrifuge for Eppendorf tubes or for 96 well plates (optional) 37 C incubator Flow cytometer Note: The FLAQ requires a flow cytometer equipped with a blue laser Notch1 excitation (488 nm) and one measurement parameter. The photomultiplicator tube (PMT) with a 525/50 nm or a 585/42 band pass filter is used for the detection Aceclofenac of FITC or RD1 (phycoerythrin) signals, respectively. == Software == Forecyt (Intellicyt) FlowJoX software (Tree Star) or other FACS analysis software == Procedure == == A. Preparation of the FLAQ beads coated with p24GagHIV-1 IIIB polyclonal antibodies == Pipet 1 ml of Sphero Protein A Polystyrene Particles (referred hereafter as beads) into an Eppendorf tube. Centrifuge for 2 min at 12, 000 rpm to pellet the beads. Remove supernatant. Add 1 ml of 1x PBS to beads pellet and centrifuge at 12, 000 rpm for 2 min. Resuspend the beads pellet with 80 l of the commercial vial of human anti-p24GagHIV-1 IIIB polyclonal antibodies sold at the concentration 1 mg/ml. Pipet three times up and down and incubate for 15 min at room temperature on a rotating mixer. Centrifuge for 2 min at 12, 000 rpm to pellet Aceclofenac the beads. Remove Aceclofenac and keep aside the antibody-containing supernatant. Add 1 ml of 1x PBS to the beads pellet. Centrifuge for 2 min at 12, 000 rpm. Add another 1 ml of 1x PBS. Centrifuge for 2 min at 12, 000 rpm. Add to the beads pellet 100 l of a 1x PBS solution containing 4. 8 mg/ml of Human normal IgG blocking reagent. Mix well by pipetting up and down 3 times. Incubate for 15 min at room temp on the rotating mixer. Centrifuge for 2 min at 12, 000 rpm to pellet the beads. Discard the supernatant. Wash the beads twice with 1 ml of 10 mg/ml of BSA in PBS, spinning 2 min at 12, 000 rpm each time. Resuspend the beads in 1 ml of 10 mg/ml of BSA in PBS and store these FLAQ beads at 4 C. Repeat steps 117 using the supernatant containing p24GagHIV-1 IIIB polyclonal antibodies from step 7 (Note 1). == B. Measuring p24Gagcontent in viral preparations == Resuspend the Abcam p24Gagprotein in FACS buffer at 8 g/ml. Aliquot and freeze. Prepare 200 l of the Abcam p24Gagprotein standard by serial dilution in FACS buffer with 0. 5% Triton-X100. Typically we perform 8 serial dilutions ranging from 10 ng/ml to 78. 12 pg/ml. Harvest viral supernatants and prepare dilutions in FACS buffer supplemented with 0. 5% Triton-X100 (Note 2). 160 l per sample will be required. Prepare a mix containing the following per sample: 0. 5 l KC57 conjugated to RD1 or FITC 0. 5 l FLAQ beads 39 l FACS buffer 0. 5% Triton X-100 Transfer 160 l of the standards and the viral supernatants to a 96 well V-bottom plate. Add 40 l of the reagent mix to each well and mix. Incubate at 37 C for 1 h. Centrifuge each plate at 2, 000 rpm for 2 min. Do not use plate sealers at this step to avoid well-to-well contamination due to the low superficial tension of the FACS buffer supplemented with 0. 5% Triton X-100. Discard the supernatants and wash beads once with 150 l of FACS buffer. Resuspend the beads in each well in 100 l of FACS buffer containing 1% PFA. Acquire samples on a flow cytometer, which will create files with an FCS extension. FCS files are flow cytometry standard files containing all the specifications needed to completely describe experimental data flow sets. Analyze the fcs files to obtain the median fluorescence intensities of the beads by the FITC or PE detectors for experiments.