Ique screener plus flow cytometer

To investigate the effect of ARTi constructs in the absence of the endogenous target gene, competitive proliferation assays were performed in several human and murine cell lines. Human HT-1080, RKO, MOLM-13, and MV4-11 cell lines were lentivirally transduced with shRNAmir expression constructs cloned into pRRL-SFFV-GFP-miRF-PGK-Neo (SGFN) backbone at 20–60% efficiency. Initial infection efficiency was determined at day 4 post transfection (day 0) by measuring GFP expression as a readout using iQue Screener Plus flow cytometer (IntelliCyt). Percentage of shRNA⁺ cells (GFP positive) was monitored by flow cytometry in regular intervals, and results were normalized to day 0.
Human GP2d, Ls513, and MIA PaCa-2 cell lines were lentivirally transduced with shRNAmir constructs cloned into pRRL-TRE3G-GFP-miRE-PGK-Puro-IRES-rtTA3 backbone (LT3GEPIR, Addgene plasmid #111177). 500 cells were seeded in duplicates in 96-well plates and treated with 1 µg ml^-1 dox for 9–10 d and analyzed with Incucyte (Sartorius). Untreated cells served as reference.
Murine NIH-3T3, EPP2, and RN2 cells were retrovirally transduced with shRNAmir constructs cloned into pMSCV-miR-E-PGK-Neo-IRES-mCherry backbone (LENC; Addgene plasmid #111163), and initial infection levels were determined by flow cytometry based on mCherry expression 4 d post transduction (day 0).

For profiling the phosphorylation activity of p190 and p210 isoforms in cell line models, a Tyrosine Phosphorylation Proarray (Full Moon Biosystems, #PST228) featuring 228 phospho-tyrosine sites was used. Sample preparation and processing were performed according to manufacturer’ instructions. Array scanning and image analysis were performed by Full Moon Bioscience and data were normalized to the median antibody signaling value. Western blotting was used to validate findings in cell lines and CML patient samples (three p190 and three p210). For Ph+ALL samples, phosphorylation of selected proteins was investigated by flowcytometry using iQue Screener Plus flow cytometer (Intellicyt). Further details are described in Supplementary materials.

The binding of PfRH5 antigen-specific antibodies to human Fc receptors (FcR) and complement C1q was measured using a previously-described assay.⁷⁴ (link)^,75 (link) Briefly, avi-tagged FCGR2A, FCGR2B, FCGR3A, and FCGR3B proteins were produced and purified by the Duke Human Vaccine Institute Protein Production Facility. These proteins were then biotinylated with BirA ligase using a commercially available kit (Avidity, #BirA500). Purified human C1q protein (Sigma, #C1740) was biotinylated using EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce, #A35358) according to the manufacturer’s instructions. These biotinylated Fc domain-binding proteins were then incubated with streptavidin-PE (Prozyme, #PJ31S) to generate the assay detection reagents. Magplex-C microspheres were coupled to biotinylated PfRH5 antigen as described above, blocked with PBSA, and added to 384-well plates so that each well contained 1500 RH5-coupled beads. Plasma from test subjects was diluted in PBSA, added to the beads, and incubated for 2 h at RT on a plate shaker (800 rpm). The beads were then washed, incubated with one of the PE/FcR conjugates for 1 h at RT on a plate shaker (800 rpm), washed again, and acquired on an Intellicyt iQue Screener PLUS flow cytometer. Results were reported as the median PE fluorescence intensity.

CAR T cells were manufactured and the ex vivo cytotoxicity assay was performed as previously described [30 (link), 31 (link)] and indicated in the Supplementary methods. The cells were stained using a designed antibodies panel (Supplementary Table 3). Cells were acquired using iQue Screener Plus flow cytometer and analyzed using the ForeCyt software (edition 6.2, Intellicyt). Details of data analysis can be found in Supplementary materials.

An assay for measuring antibody-dependent THP-1 monocyte / cellular phagocytosis (ADCP) was used as previously described.⁶⁸ (link) Briefly, 1 μm yellow-green fluorescent NeutrAvidin beads were coupled to biotinylated PfRH5 antigen and blocked overnight with PBSA. The beads were then washed twice with PBSA, diluted to 1.8 × 10⁸ beads/mL, and 10 μL beads/well were added to a 96-well round-bottom microplate. Diluted plasma from immunized subjects (10 μL/well) was added to the beads and incubated at 37°C for 2 h to allow the formation of immune complexes. Unbound antibodies were washed off, then 25,000 THP-1 cells/well (ATCC, #TIB-202) were added to the beads in 200 μL THP-1 medium (R-10 + 55 μM β-ME) and incubated overnight at 37°C. Cells were fixed and acquired on an Intellicyt iQue Screener PLUS flow cytometer. The phagocytic score for each sample was calculated as (% bead-positive cells) x (gMFI of bead-positive cells)/(10 x gMFI of first bead-positive peak).