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Fig. 2 | Arthritis Research & Therapy

Fig. 2

From: Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue

Fig. 2

High synovial cell yield, preserved surface markers, and reproducible transcriptomic results with mechanical and enzymatic disaggregation protocol. a Total cell counts per gram from synovial tissue mechanically disaggregated with or without Liberase™ TL proteolytic enzyme treatment. n = 16, paired t test. b, c Flow cytometry detection and quantification of stromal cells (CD45PDPN+) upon mechanical disaggregation with or without Liberase™ TL proteolytic enzyme treatment. d Flow cytometry of dissociated synovial cells treated with panel of proteolytic Liberase™ TL formulations. Cells gated for viability and CD3 T-cell receptor subunit. Plots representative of n = 4 biologic replicates. e Total RNA yield from synovial tissue dissociated with three concentrations of Liberase™ TL. Two or three technical replicates from nine tissues used for each enzyme concentration. ANOVA with Tukey's comparison, *p<0.05 and **p<0.01. f Principal component analyses on RNA-seq gene expression results for samples from (e), whereby each technical replicate of same sample is represented by a dot of same color while variation between replicates is indicated by size of encircling cloud of same color. g Sum of squares within (SSW) replicates from same donor divided by total sum of squares, using gene expression profiles from (e) and (f). Horizontal lines indicate SSW values using all replicates. Individual dots are SSW values computed after leave-one-out strategy where replicates from one donor are left out and SSW is computed with remaining samples. Student’s t test used to test whether bootstrapped SSW values differ significantly. DH Dispase-High, DL Dispase-Low, PC principal component, PDPN podoplanin, TH Thermolysin-High, TL Thermolysin-Low, TM Thermolysin-Medium

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