Counting beads

Viable cell numbers were determined by flow cytometry using counting beads (Life Technologies, Grand Island, NY). Apoptosis was estimated via flow cytometry measurement of phosphatidylserine externalization with annexin V staining (BD Biosciences, San Jose, CA). Cell membrane integrity was simultaneously assessed by 7-aminoactinomycin D (7AAD) exclusion in the annexin V-stained cells. To assess cell numbers and apoptosis in leukemia cells co-cultured with MSCs, CD45⁺ cells were counted and apoptotic cells were defined as annexin V⁺/AAD⁺ CD45⁺ cells.

Viable cell numbers were determined by flow cytometry using counting beads (Life Technologies, Grand Island, NY). Apoptosis and cell membrane integrity were simultaneously assessed by flow cytometric analysis of annexin V (AnnV) (BD Biosciences, San Jose, CA) and 7-aminoactinomycin D (7AAD) (Sigma, St. Louis, MO). For leukemia cells co-cultured with MSCs, CD45⁺ cells were counted, and apoptotic cells were defined as AnnV+/7AAD+ CD45⁺ cells. For primary samples, AnnV positivity was determined in bulk (CD45⁺) and CD34⁺CD38⁻/CD34⁺CD38⁺ leukemia stem/progenitor cells or CD34⁺ normal BM cells.

Viable cell numbers were determined by flow cytometry using counting
beads (Life Technologies, Grand Island, NY). Apoptosis was estimated via flow
cytometry measurement of cells after Annexin V staining (BD Biosciences, San
Jose, CA) in the presence of 7-aminoactinomycin D (7AAD). For AML and MSC
co-culture experiments, CD45⁺ cells were counted and apoptotic cells
were defined as Annexin V⁺/7AAD⁺CD45⁺. For
primary samples, apoptosis was assessed in both CD45⁺ bulk and
CD45⁺CD34⁺ stem/progenitor cells and expressed as
specific apoptosis: $$\frac{\%\text{of}\text{apoptosis}\text{in}\text{treated}\text{cells}-\%\text{of}\text{apoptosis}\text{in}\text{untreated}\text{cells}}{\%\text{of}\text{viable}\text{untreated}\text{cells}}\times 100\%$$

All biotinylated and fluorescent antibodies, human CD138 microbeads, and Pan T Cell Isolation Kits were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). DMEM, RPMI-1640, L-glutamine, penicillin-streptomycin, and phosphate buffered saline (PBS) were purchased from Corning (Corning, NY). Fetal bovine serum, live-cell dyes, lipophilic tracers, collagenase, and counting beads were purchased from Life Technologies (Carlsbad, CA). 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (DSPE-PEG2000), and polycarbonate membranes were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol and chloroform were purchased from Millipore Sigma (Burlington, MA). Streptavidin conjugation kit was purchased from Abcam (Cambridge, United Kingdom). Human Cytokine Array Q1 was purchased from Raybiotech (Peachtree Corners, GA). All mice used in this study were NCG (strain: 572), female, 50–56 days old, and purchased from Charles River (Wilmington, MA). All mice experiments in this study was in compliance with the Institutional Animal Care and Use Committee at Washington University.

Human cancer cells genetically modified to express EGFP were seeded in 12-well plates and infected with 1,000 viral particles (vp) per cell of HDAds or treated with 10 μg/mL of anti-human PD-L1 IgG, Isotype IgG (Biolegend). HER2.CAR T-cells with an effector to target ratio of 1:20 were added 48 hours post-infection and cultured for 5 additional days. Residual live EGFP⁺ cancer cells and T-cells were counted on the basis of EGFP and CD3 expression with Counting Beads (Life Technologies). Cell numbers were calculated per 5,000 microbeads.

LSK cells were sorted as described above and cells were placed in StemSpan SFEM serum free media (Stem Cell Technology, Vancouver, BC, Canada) supplemented with 100 ng/ml SCF (Peprotech). Five hundred cells per well in multiple wells on a 96 well plate were seeded on day 0 with feeding of fresh media and SCF occurring every two days. On days 3, 6, 9, 12, and 15, cells were harvested in quadruplicate, stained for lineage and hematopoietic stem cell surface markers as described above and cells were acquired on the FACS Aria III. Cell counts were obtained flow cytometrically using counting beads (Life Technologies) according to the manufacturer's instructions.