(551.3) Evans Blue Dye Interferes with Gating for Certain Fluorochrome-Conjugated Antibodies Using Flow Cytometry in the Ischemic Rat Heart
Sunday, April 3, 2022
10:15 AM – 12:15 PM
Location: Exhibit/Poster Hall A-B - Pennsylvania Convention Center
Poster Board Number: E40
Jiajing Teng (Penn State University), Cesar De Jeronimo Diaz (Penn State University), Ryan Flynn (Penn State University), Matthew Lin (Penn State University), Connie Rogers (Penn State University), Donna Korzick (Penn State University)
Background: Acute myocardial infarction (AMI) is the leading cause of death in the U.S. Understanding the immune response to AMI has potential therapeutic impact. In particular, the differentiation of monocytes into macrophages post-AMI is of interest given their role in inflammatory cytokine production and tissue repair. Recent studies suggest that impaired macrophage (Mϕ) polarization may promote a proinflammatory phenotype in the heart post-AMI. Flow cytometry is a gold standard technique to assess immune cell populations including M1 and M2 Mϕ subtypes, using a wide range of fluorochrome-conjugated antibodies (FC-Abs). Evans Blue (EB) dye is widely used to assess the size of AMI, but the feasibility of the combining approaches due to spectral overlap is uncertain. Thus, the objective of this study was to determine whether the cardiac viability dye EB can be used simultaneously with FC-Abs to quantify M1 and M2 Mϕ in cardiac tissue post-AMI.
Methods: Immune cells were isolated from ischemic rat hearts (31 min ischemia/48 hr reperfusion) with or without EB perfusion and stained with FC-Abs (1 μg/1 × 106 cells) to the following cell surface markers: FITC-CD45 (experiment 1) or AF750-CD45 (experiment 2) to detect leukocytes; Pacific Blue-CD11b, AF700-CD68 and PE-CD206 (experiment 1) or Pacific Blue-CD11b, AF488-CD68 and PE-CD206 (experiment 2) to quantify M1 and M2 Mϕ. A total of 500,000-1,000,000 events were acquired using a BD LSR-Fortessa and were analyzed and plotted using FlowJo software v10.
Results: Overlap of EB (emission peak at 680 nm) was detected in the AF700-CD68 channel (emission peak at 720nm) and the AF750-CD45 channel (emission peak at 775nm), interfering with gating within these two channels. In experiment 1, the M1 (CD45+CD11b+CD68+) population differed between samples with EB (2.5% of CD45+CD11b+ population, sample size n=2) vs without EB (68.5%, n=2). However, in experiment 2, the M1 population was similar between samples with EB (54.3% of CD45+CD11b+ population, n=2) and without EB (48.7%, n=1). The M2 (CD45+CD11b+CD206+) population did not differ in experiment 1 or 2.
Conclusion: Our data suggest that the EB perfusion interferes with fluorochromes with emission peaks close to 680nm. Thus, caution must be taken when selecting FC-Abs for flow cytometry when combined with EB perfusion or other related dyes. Our next steps will be to confirm these observations in a larger sample size.
