True nuclear transcription buffer set

Weighted tumors were cut into pieces and digested in a mixture comprising collagenase IV (1 mg mL^–1), hyaluronidase (0.4 mg mL^–1), DNase I (0.1 mg mL^–1), and bovine serum albumin (10 mg mL^–1) in RPMI‐1640 medium at 37 °C under constant shaking for 30 min. Debris was removed by filtration through a 40 µm mesh. After lysing red blood cells, the cell mixture was washed and resuspended in PBS supplemented with 2% FBS for further analyses.
Spleens were minced, filtered through 40 µm meshes, and subjected to red blood cell lysis to obtain suspension of splenocytes. The splenocytes or digested tumors were stained with Zombie NIRTM Fixable Viability Kit (Biolegend, USA), blocked with antimouse CD16/32, and then stained with the antibodies listed in Table S3, Supporting Information. Intracellular staining was performed after fixation and permeabilization with a Biolegend True‐Nuclear Transcription Buffer Set. Samples were analyzed on a BD FACSVerse 3L8C cytometer (San Jose, CA, USA). Data were processed with FlowJo software V10 (BD Biosciences, San Jose, CA, USA).

Fetal liver cells were processed into a single‐cell suspension and cKit‐enriched using CD117 MicroBeads (Miltenyi Biotec, San Diego, CA, USA). The cKit‐enriched population was stained with an antibody cocktail for surface markers of hematopoietic stem and progenitor cells. Cells were then fixed and permeabilized with the True‐Nuclear Transcription buffer set (Biolegend) and then stained with Ki67‐APC (Invitrogen, Carlsbad, CA, USA) and Hoescht 33,342 (Invitrogen).