Mouse lineage cell depletion kit

Samples were pre-incubated with Fc Block (eBioscience) for 10 min. Staining of surface molecules with fluorescently labeled antibodies was performed on ice for 30 min in the dark. For phenotypic analysis by flow cytometry, samples were run on an LSR Fortessa (BD Biosciences) and data were analyzed using FlowJo (Tree Star). For sorting of hematopoietic progenitors or ILC2s from bone marrow cells, lineage negative (Lin⁻) cells were firstly enriched by mouse Lineage Cell Depletion Kit (Miltenyi Biotec) following the manufacturer’s instructions. Cells were sorted on an Aria II (BD Biosciences).

Lin⁻ cells were purified from the bone marrow of three mice 16 weeks after transplantation using immuno-magnetic bead selection with the Mouse Lineage Cell Depletion Kit (Miltenyi, 130-090-858). For each animal, 500 Lin⁻ cells were plated in triplicate in methylcellulose (STEMCELL Technologies, Methocult GF M3434) incubated at 37 °C. After 12 days, 30-35 colonies per mouse were picked and washed in PBS before prior to lysis. HTS analysis was performed on all colony lysates and allelic editing for each colony was classified based on editing percentages.

Hematopoietic progenitor cells (i.e., Lin^-) were obtained from a cohort of 6-weeks-old B6.SJL (CD45.1) mice five days after 5-FU treatment (150 mg/kg) using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec Inc., Auburn, CA), and were then co-transduced with MSCV-PIG+MSCVneo (negative control), MSCV-PIG+MSCVneo-MLL-AF9 (positive control), MSCV-PIG-miR-26a+MSCVneo-MLL-AF9, and MSCV-PIG-miR-29a+MSCVneo-MLL-AF9 through “spinoculation”. Then, two aliquots of 2x10⁴ of the infected cells were plated into two 35 mm Nunc petri dishes in 1.5 ml of Methocult M3231 methylcellulose medium (Stem Cell Technologies Inc., Vancouver, BC, Canada) containing 10 ng/ml each of murine recombinant IL-3, IL-6, and GM-CSF (R&D Systems, Minneapolis, MN) and 30 ng/ml of murine recombinant stem cell factor (Sandoz, Holzkirchen, Germany), along with 1.0 mg/ml of G418 (Gibco BRL, Gaithersburg, MD) and 2.5 μg/ml of puromycin (Sigma, St. Louis, MO). Cultures were incubated at 37° C in a humidified atmosphere of 5% CO₂ in air for 6–7 days. Cells were collected and replated with two aliquots of 2x10⁴ of colony cells into two dishes every 7 days up to three or four passages.

HL-60 cells (ATCC CCL-240) were obtained from the American Type Culture Collection (Manassas, VA) and maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) with 20% FBS. HL-60 cells were incubated in IMDM +20% FBS with and without 50 ng/ml SDF-1 (R&D, Minneapolis, MN) for two and 24 hours, respectively. This concentration of SDF-1 has been shown to elicit optimal responses in numerous of our assays [12 (link),18 (link),19 (link),21 (link),41 (link)]. C57Bl/6 strain mice were used to isolate lineage negative bone marrow cells. The Indiana University Committee on Use and Care of Animals approved the mouse studies. Mouse lineage negative cells were isolated using the Miltenyi Biotech (San Diego, CA) mouse Lineage Cell Depletion Kit. After lineage depletion, lineage negative cells were incubated in IMDM +10% FBS and stimulated with or without 50 ng/ml SDF-1 (R&D, Minneapolis, MN) for two and 24 hours, respectively.