Optimized Protocols for Isolation, Fixation and Flow Cytometric Characterization of Leukocytes in Ischemic Hearts

Supplemental Figure 1. SSC-A (granularity) and FSC-A (size) of unfixed splenocytes and splenocytes fixed either with 1%PFA or ice-cold methanol or acetone. Data were analyzed using repeated measures 1-way ANOVA with Dunnet’s multiple comparisons test, and **p value <0.01 and ***p value <0.001 was considered significant (n=5 in each group).

Supplemental Figure 2. (A) Representative flow histograms for circulating CD45⁺ leukocytes stained with intravascularly injected anti-CD45 BV605 antibody. (B) Representative flow cytometry scatter plots for anti-CD45 BV605 staining in the cells isolated from the spleen (top) and mediastinal lymph nodes (bottom), and (C) their quantitative group data. (D) Group quantitative data for total CD45⁺ leukocytes in the supernatants obtained after 50g and 350g centrifugation of minced cardiac tissues. Data were analyzed using unpaired, 2-tailed, student’s ‘t’ test and a *p value <0.05 was considered significant (n=4-5 in each group).

Supplemental Figure 3. (A) Quantitative group data for CD45⁺CD4⁺, CD45⁺CD4^-CD11b⁺Ly6G⁺, CD45⁺CD4^-CD11b⁺Ly6G^-Ly6C⁺ (Ly6C^Lo and Ly6C^Hi) and CD45⁺CD4^-CD11b^-Ly6G^-Ly6C^-B220⁺ cells in blood-borne CD45 BV605⁺ cells observed in cardiac digests of perfused and non-perfused hearts. Data for each cell type between the perfused and non-perfused groups were analyzed using unpaired, 2-tailed, student’s ‘t’ test and a *p value <0.05 was considered significant (n=5 in perfused and 4 in non-perfused group).

Supplemental Figure 4. Representative flow cytometry scatter plots for 7-AAD staining in cardiac digests. Aliquots from one digested heart (as representatives) from each group were analyzed by live/dead staining to ensure >95% cell survival during the isolation process.