Cyan flow cytometer

Cells were harvested and washed in ice-cold PBS supplemented with 1% (v/v) FBS and 0.08% sodium azide. Cellular debris were excluded based on size (forward scatter [FSC]) and granularity (side scatter [SSC]). The FSC/SSC gate for Monocyte comprised ∼60%, total events. Couplets were excluded based on SSC versus FSC and SSC versus pulse width measurements. Samples were stained for cell surface markers with FITC, PE, and APC conjugated antibodies. For polarization analysis, human antibodies for CD23, MHCII (both M1 marker), CD163, CD206, and dectin (M2 marker) were purchased from BD Pharmingen or BioLegend. Unstained and isotype control treated cells were used as controls. Samples were analyzed using a FACScan or BD Cyan flow cytometer using CellQuest software (BD Biosciences, San Jose, CA. Further analysis was performed using FlowJo software (Tree Star, Ashland, OR). Cells were gated according to their forward scatter (FSC) and side scatter (SSC) properties including the larger cells with high granularity and excluding the small-sized debris with a low SSC and FSC shown at the bottom left corner of the dot plot.

Calu-3 cells were incubated in 10 μM bromodeoxyuridine (BrdU) (Sigma) for 60 min at 37°C. Cells were then washed, and trypsinised before fixation in ice-cold ethanol. Cells underwent 30 min digestion in warmed pepsin solution (Sigma), before treating with 2M HCl for 15 min. Samples were washed, then blocked (0.5% BSA/0.5% Tween 20) for 30 min before staining with a-BrdU-488 (Biolegend) directly conjugated antibody and Propidium Iodide (Invitrogen). Samples were acquired on BD Cyan Flow Cytometer and analyzed via Flow-Jo.

Vector titration by flow cytometry: 1 × 10⁵ HEK293T cells were plated into each well of a 6-well plate and transduced with a range of volumes of concentrated lentivirus. At 72 hr after transduction, cells were trypsinized and EGFP-positive cells were quantified using a BD Cyan flow cytometer or BD FACSArray Bioanalyzer 3 days after transduction.
HEK293T cells were not used for qPCR titration due to their reported abnormal karyotype.⁵⁶ (link) Briefly, 1 × 10⁵ HT1080 cells were plated into each well of a 6-well plate and transduced with a range of volumes of concentrated lentivirus. At 72 hr after transduction, genomic DNA was extracted and the provirus titer calculated by qPCR, as described previously.⁵⁷ (link) The viral capsid number was determined using a p24 ELISA kit (Clontech - 632200). The capsid number was determined according to the kit manufacturer’s calculations, where 1 ng p24 is equivalent to 1.25 × 10⁷ lentiviral particles (lp). The vector RNA genome titer was determined using a qRT-PCR RNA titration kit containing pre-designed primers and standards (Clontech - 631235). For all titer comparisons, LTR1 and third generation vectors were produced side-by-side to account for variations between production batches.