Countbrighttm absolute counting beads

A375 and WM1361 cells were seeded at 2 × 10⁵ cells per well in a 6-well plate and treated with 100 µl of 100 nM anti-CSPG4-PDD and incubated for 96 hours. Supernatants and detached cells were washed once in 1× Binding Buffer (included in Annexin V Apoptosis Detection Kit APC, 88-8007-72, eBioscience) and resuspended in 2 mL. Then, 1 mL of the suspension was used per sample, centrifuged and 5 µL of APC-conjugated Annexin V were added to 100 μL of cell suspensions and cells were then incubated for 10–15 minutes at room temperature. Cells were washed in 1× Binding Buffer and resuspended in 200 µL of 1 x binding buffer. Cells were then treated with 100 μL of DAPI (D1360, Thermo Fisher Scientific) staining solution (1:10,000) and 10 µL of CountBright^TM Absolute Counting Beads (C36950, Invitrogen^TM, Loughborough, UK) and immediately analyzed on a FACSCanto II^TM flow cytometer. Cells were then analyzed by gating early apoptosis (Annexin+, DAPI-), late apoptosis (Annexin+, DAPI+), necrosis (Annexin-, DAPI+) and as well as live cells (Annexin-, DAPI-).

Highly synchronous NF54 P. falciparum trophozoites were purified by MACS on 25 LD columns (Miltenyi Biotec) and supplied with fresh media. At the late-pigmented trophozoite to early schizont stage, the cysteine-protease inhibitor E64, 10 μM, was added for 8–12 h to allow schizont maturation without rupturing. Merozoites were released from mature schizonts by passing the culture through a 1.2 μM pore size filter (Pall GmbH) that was pre-blocked with 1% casein-PBS. The filtrate was collected and centrifuged at 4,000 g for 15 min and resuspended in 5 ml culture media. The cell density was determined by staining merozoites with dihydroethidium (DHE) for 30 min in the dark and subsequently mixed with CountBrightTM Absolute Counting Beads (Invitrogen) at a 1:100, 1:50 and 1:25 dilution. The merozoite concentration of the three dilution was quantified using the formula: the difference of merozoite count between stain and unstained condition/count of beads × dilution factor × number of beads.

All oocyst samples purified from mouse intestine were incubated in a final concentration of 1% paraformaldehyde for 15–30 min prior to analysis. Briefly, 40 ul of sample and 50 ul of CountBright^TM absolute counting beads (Invitrogen, Burlington, ON, Canada) were added to a paraformaldehyde solution for a final volume of 500 ul. Oocysts within each sample were quantified by morphology (FSC-A and SSC-A) using BD LSRFortessa^TM cell analyser (BD, Franklin Lakes, NJ, USA). Oocysts were initially identified with a FITC-labeled mouse IgG3 monoclonal antibody targeting a surface antigen (AbD Serotec, Raleigh, NC, USA). Results demonstrated that 98% of the FITC positive events occupied a distinct population by size and complexity in the FSC-A and SSC-A channels. Results generated by flow cytometry are based on unstained samples gating on this distinct oocyst population. Oocyst concentration per sample was calculated by determining the quantity of sample collected based on the number of CountBright^TM bead events collected. Final oocyst numbers were normalized by gram of mouse intestines.

CellTrace Violet (Invitrogen, ThermoFisher Scientific, Germany) labeled KG1a, MOLM-13, HL-60, ML-2, SUPB15 cells, or primary AML cells (target cells) were used for the T cell-mediated cytotoxicity assay. In brief, anti-CD123 CAR T cells (effector cells) were incubated with target cells at the indicated ratios for 16 h in RPMI-1640 medium (supplemented with 20% FCS, 2 mM glutamine, 100 U/mL penicillin/streptomycin). Percentage-specific lysis of the target cells was determined using flow cytometry. Cells were harvested following the incubation period, stained with 7-AAD (BD Biosciences, Germany) and 10 μL CountBright^TM absolute counting beads (Invitrogen, ThermoFisher Scientific, Germany) were added to each sample in a fixed volume just prior to flow acquisition. A uniform number of bead events were acquired for each sample on the flow cytometer. Residual live target cells were CellTrace^TM violet⁺ 7-AAD-. Unstained and FMO controls were used to identify gating boundaries.

Bone marrow from hind legs and hip bones were flushed in a fixed volume of PBS (20 mL). Cells from the BM of each mouse were counted based on the flushing volume and equally distributed in the FACS tubes for staining. A fixed number of bead events were acquired during FACS analysis along with a fixed volume for acquisition (150 μL). Absolute cell counts were calculated according to the formula provided by the CountBright^TM Absolute counting beads manufacturer’s protocol (Invitrogen, Germany).

Two percent sodium alginate was prepared by dissolving sodium alginate (Sigma-Aldrich) in phosphate-buffered saline (PBS) and sterilizing by heating to 80°C in a water bath for 15 min (Leo et al., 1990 (link)). Anti-CD150-PE, anti-GRPC5C Alexa Fluor-405, and anti-Ki67-Alexa Fluor 405 antibodies for flow cytometry were purchased from R&D Systems. Anti-CD49f-PE and anti-CD90-VioBlue were purchased from Miltenyi Biotec. Transforming growth factor beta-1 (TGFβ-1) and fibroblast growth factor were obtained from PeproTech. Fms-like tyrosine kinase-3 (Flt-3), G-CSF, stem cell factor (SCF), and interferon alpha were purchased from ImmunoTools. All-trans retinoic acid (ATRA) was obtained from Sigma-Aldrich. Hoechst 33342, propidium iodide (PI), and pyronin Y (PY) were purchased from Sigma-Aldrich. The CountBright^TM Absolute Counting Beads was purchased from Thermo Fisher Scientific. MHY1485, AZD8055, rosiglitazone, KU-55933, LEE011, roscovitine (seliciclib, CYC202), glasdegib (PF-04449913), sodium butyrate, dasatinib, BIO, plerixafor (AMD3100), quizartinib, and sorafenib were purchased from Selleckchem. Adiponectin, triglitazone LE135, PD169316, harmine, nilotinib, ethylisopropyl amiloride, anisomycin, and curcumin were purchased from Sigma-Aldrich, while prostaglandin E2 was from BioVision/Cambridge Bioscience. Cytarabine and daunorubicin were purchased from Sigma-Aldrich.