Gelatin coated glass bottom 24 well plates

HOS CXCR4-2A-CD4 were seeded onto gelatin coated glass bottom 24 well plates (MatTek) and infected at an approximate MOI of <1 with HIV-1_WT, L_CG-HI, or L_GC-HI. Twenty-eight hours after infection the cells were washed with PBS and fixed with 4% paraformaldehyde (Thermo) in PBS for 30 min at RT. After permeabilization with 70% ethanol for 2hr at RT the cells were briefly washed with Stellaris RNA-FISH wash buffer A for 5 min at room temperature. The cells were then incubated with custom Stellaris smFISH probes targeting HIV-1 gag or all viral mRNAs (Biosearch Technologies) at a concentration of 0.125 μM in Stellaris RNA FISH hybridization buffer for 16–18 hr at 37°C. The cells were then washed two times for 30 min at 37°C in Stellaris RNA FISH wash buffer A. The second wash contained Hoechst dye at 1 μg/ml. After a 5 minute wash with Stellaris RNA FISH wash buffer B cells were rinsed three times with PBS and imaged by deconvolution microscopy (Deltavision). Image stacks were generated by maximum intensity projection using the Z project function in ImageJ (Version 2.0.0-rc-59/1.51n). RNA spots were counted using Find Maxima function in ImageJ.