Cy5 conjugated anti mouse secondary antibody

Cells were collected by trypsinization, fixed with 70% ethanol overnight at 4 °C and washed three times with PBS. The fixed cells were incubated with 0.25% Triton X-100 in PBS for 15 min and washed with PBS three times. Next, the cells were stained with phospho-MPM2 (Millipore, #05-368, RRID:AB_309698, 1:500) antibody for 1 h at room temperature and washed three times with PBS. Then, the cells were incubated with Cy5-conjugated anti-mouse secondary antibody (Life technologies, A10524, RRID:AB_2534033, 1:2000) for 30 min and washed three times with PBS. Cells were then suspended in Propidium Iodide (PI)/RNase Staining Buffer (BD Pharmingen) and analysed by flow cytometry (LSR-II, BD Biosciences). FlowJo software (Version 10.0) was used for data analysis (Tree Star, Ashland, OR).

Cells were collected by trypsinization, fixed with 70% ethanol overnight at 4 °C and washed three times with PBS. The fixed cells were incubated with 0.25% Triton X-100 in PBS for 15 min and washed with PBS three times. Next, the cells were stained with phospho-MPM2 (Millipore, #05-368, RRID:AB_309698, 1:500) antibody for 1 h at room temperature and washed three times with PBS. Then, the cells were incubated with Cy5-conjugated anti-mouse secondary antibody (Life technologies, A10524, RRID:AB_2534033, 1:2000) for 30 min and washed three times with PBS. Cells were then suspended in Propidium Iodide (PI)/RNase Staining Buffer (BD Pharmingen) and analysed by flow cytometry (LSR-II, BD Biosciences). FlowJo software (Version 10.0) was used for data analysis (Tree Star, Ashland, OR).

TMAs were blocked with background Buster solution (Innovex) for 30 min, followed by avidin–biotin for 8 min. Human FITC-Lineage (CD2, CD3, CD14, CD16, CD19, CD56, CD235a), KLRG1 antibodies were applied at 4 °C overnight and followed by a Cy5-conjugated anti-mouse secondary antibody (Thermo Fisher Scientific, CA, USA). For mice, mouse FITC-Lineage (CD3, CD45R, CD11b, CD19, Ly-G6), GATA3 (Thermo Fisher Scientific, CA, USA), CD127 (Thermo Fisher Scientific, CA, USA) antibodies were applied at 4 °C overnight and followed by a PE-conjugated anti-rat (Thermo Fisher Scientific, CA, USA) and a Cy5-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific, CA, USA) according to the manufacturer's instructions. After staining, counterstained the TMAs with DAPI (Sigma Aldrich) for 10 min. For quantification, each nucleus was segmented using the DAPI channel after proper processing and background subtraction. Counter the number of cells with a particular combination of markers. KLRG1 expressing Lineage⁻ cells were defined as Lineage⁻ KLRG1⁺ nucleated cells. For each patient, the frequency of Lineage⁻ KLRG1⁺ was calculated in triplicate cores, and then the average frequency of triplicate cores was measured to calculate the final cells.