Facsarray bioanalyzer

Human CBMCs and PB MCs (PBMCs) were incubated with lipopolysaccharide (LPS) for 48 h at 37°C before supernatants were collected. Under the same time and temperature conditions, CBMCs were also cultured in the presence of either anti-IL-10 (IL-10 neutralizing) antibody or IL-12 alone or with LPS. Cytokine levels were measured in supernatants using Cytometric Bead Array Flex Sets and analysed on BD FACS Array Bioanalyzer (BD Biosciences, Franklin Lakes, NJ, U.S.A.) [30 (link)].

Blood was collected by venipuncture from a peripheral vein into BD Vacutainer SSTII Serum Separator Tubes (cat#367955). The tubes were gently inverted 5–6 times, left to clot for 30 min at room temperature and centrifuged for 10 min at 1600× g at RT. The serum was aliquoted and stored at −70 °C until needed.
Measurement of the concentration of the cytokines IL-2, IL-4, IL-6, IL-10, IL-17a, IFNγ and TNF in patient serum samples was performed with a BD FACS Array Bioanalyzer, using a cytometric bead array (CBA) assay (Human Th1/Th2/Th17 Kit, cat#560484, BD Biosciences, San Diego, CA, USA) (range 20–5000 pg/mL for all analytes, sensitivity 2.6 pg/mL for IL2, 4.9 pg/mL for IL4, 2.4 pg/mL for IL6, 4.5 pg/mL for IL10, 18.9 pg/mL for IL17a, 3.7 for IFNγ and 3.8 pg/mL for TNF).

Unless stated otherwise, 100,000 cells were analyzed for EGFP expression in a BD FACSArray Bioanalyzer. During analysis, live cells were determined by gating forward light scatter versus side scatter and isolating the relevant population. EGFP⁺ cells were determined by plotting EGFP fluorescence (detected using a 530/30 nm bandpass filter) versus emission from the yellow channel (detected using a 575/26 band-pass filter) to compensate for auto-fluorescence. Non-transduced controls were used to gate background expression in each channel. All flow cytometry data were analyzed by FlowJo software version 9.3.1 (Tree Star).

Cells were trypsinised and 200 μl of the suspension was added to a round bottom 96-well-plate for analysis in a BD FACSArray™ Bioanalyzer. GFP fluorescence was excited with a 488 nm argon laser. During analysis of cytometry plots, live cell populations were gated by plotting forward-light-scatter versus side-scatter to visualise and isolate the viable population. GFP-positive populations were determined by plotting the emission from the green channel (detected using 530/30 nm band pass filter) against emission from the yellow channel (detected using 575/26 band pass filter), to compensate for auto-fluorescence events. Unless mentioned otherwise, non-transduced populations were used to set the baseline for GFP expression.
During NIGW investigations, homozygous 3′NIGW vectors (without a recognised promoter) expressed low-level GFP, which was presumably driven by an IRES-mediated promoter trap^{51 (link), 52 (link)}. For this reason, the baseline for GFP was gated against a homozygous control (5′wtDIS + 3′wtDIS sample) to compensate for any IRES-driven expression from unrecombined proviruses. The gated GFP-positive cell populations were used to estimate the amount of reconstituted, full-length proviruses driven by the SFFV promoter.
Where mentioned, GFP-positive cells were sorted on a MoFlo sorting machine.
All FACS data were analysed by FlowJo software version 9.3.1 (©Tree Star, Inc).

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.

Serum and CSF samples from patients and controls were collected and stored at −75°C until processing. Determination of serum and CSF levels of the cytokines IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, and IL-17A was performed on a BD FACSArray Bioanalyzer using the Cytometric Bead Array (CBA) assay (human Th1/Th2/Th17 Cytokine Kit, BD Biosciences, San Diego, USA). Serum and CSF levels of TGF-β1 were measured by ELISA (R&D Systems Quantikine TM, Minneapolis, MN, USA). The data were analyzed using the CurveExpert V1.4. Cytokine ratios (Th1/Th2, Th1/Th17, Th17/Th2, Type-1/Type-2, IFN-γ/IL-10, and IL-17A/IL-10) were also calculated (Table 1).

Apoptosis in bulk and CD34⁺CD38⁻ cells was estimated by flow cytometric measurements of annexin V stained cells using a BD FACSArray Bioanalyzer. CFSE stained cells were incubated with CD34 antibody and 7-amino-actinomycin D (7AAD). Apoptosis was estimated in 7AAD⁻ CML progenitor cells using a BD LSR-II flow cytometer. Apoptotic quiescent or proliferating CML progenitor cells were defined as annexin V positivity in the CD34⁺CFSE^bright or CD34⁺CFSE^dim population. Apoptotic leukemia cells co-cultured with MSCs are defined as annexin V+/CD45⁺ cells. Apoptosis was expressed as specific apoptosis [14 (link)]:
$\begin{array}{ccc}\frac{\%\text{\hspace{0.17em}\hspace{0.17em}of\hspace{0.17em}apoptosis\hspace{0.17em}in\hspace{0.17em}treated\hspace{0.17em}cells}-\%\text{\hspace{0.17em}of\hspace{0.17em}apoptosis\hspace{0.17em}in\hspace{0.17em}untreated\hspace{0.17em}cells}}{\%\text{\hspace{0.17em}of\hspace{0.17em}visible\hspace{0.17em}\hspace{0.17em}untreated\hspace{0.17em}cells}}& \times & 100\%\end{array}$