Figure 2.

Experimental protocol workflow

(1) On three acquisition days, samples for each batch were thawed (here represented by Day1) in the cold room on hula mixer, for approximately 40 min. After thawing, lysis buffer was added to each tube (for the purpose of figure clarity magnifier shows one tube, though all samples were processed in parallel). Next, samples were filtered through a 100 μm strainer and transferred to a 15 mL tube containing lysis buffer. Tubes were washed with additional 1 mL of lysis buffer and 15 mL tubes were filled with lysis buffer. Samples were placed on hula mix and lysed for 10 min at RT. Next, they were centrifuged and the supernatant was decanted by inversion. Cells were washed with CSB followed by one wash with DPBS. Then, they were counted, aliquoted at 1.5∗10⁶ cells/mL and centrifuged. (2) Cells were barcoded using palladium-based barcoding (colored tubes represent different barcodes) and washed 3 times with CSB. After last wash, cells were pooled together into a single tube. Next, surface staining was performed, followed by 2 washing steps with Perm-S and intracellular staining. Afterward, cells were washed and stained with Ir, washed again and kept in PFA o/n at 4°C. (3) Samples were acquired on CyTOF, generating Aliquot.FCS files. Shortly, every aliquot of ∼200 μL was washed once with CSB and two times with MilliQ water. Before the last wash, 10 μL of cell suspension was used for cell counting. Cells were resuspended in bead solution at 0.8∗10⁶ cells/mL and acquired at an event rate below 400 events/s. This step was repeated until desired number of cells was acquired and the protocol was repeated on the following days for Day2 and Day3.