alexafluor 647  (Bio-Rad)

Journal: Frontiers in Cardiovascular Medicine

Article Title: A porcine model of acute rejection for cardiac transplantation

doi: 10.3389/fcvm.2025.1549377

Figure Lengend Snippet: A porcine model of acute rejection for cardiac transplantation. (A) Yucatan pigs are first genotyped for SLA haplotype. Donor-recipient pairs are selected where there is complete mismatch of all SLA class I and class II genes. The recipient pig begins receiving immunosuppression therapy three days prior to transplantation. (B) The heart is procured from the donor pig and undergoes ex vivo normothermic machine perfusion for two-hours. The recipient pig then undergoes abdominal heterotopic heart transplantation. Following transplantation, the recipient continues to receive immunosuppression therapy for 14-days, at which time (C) close observation begins for monitoring acute rejection driven primarily by cellular processes involving CD4 + and CD8 + cells. (D) Assessments through ultrasound, CMR, histology, blood analysis, and cfDNA quantitation were used to monitor for acute rejection. (E) Observation was continued until fulminant rejection was reached.

Article Snippet: OCT embedded frozen 10 μm thick sections were stained for both CD3ɛ (Invitrogen MA1-90582, mouse, 1:150) to identify the presence of T cells, and CD8α (BioRad MCA6048GA, rabbit, 1:150) to identify the subset of cytotoxic T cells.

Techniques: Transplantation Assay, Ex Vivo, Quantitation Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: A porcine model of acute rejection for cardiac transplantation

doi: 10.3389/fcvm.2025.1549377

Figure Lengend Snippet: Histologic and functional changes of acute rejection. (A) Palpation grade was assessed 3–4 times daily for each animal during the observation period (peri-operative immunosuppression period shaded in blue). (B) Troponin levels were measured 2–3 times a week and were significantly greater at the time of fulminant acute rejection relative to POD 19-23 and baseline (Wilcoxon matched-pairs signed rank test). (C) Echocardiography was used as an adjunct to palpation grading to assess for cessation of cardiac activity and qualitative trending of LV wall thickness. (D) Cross sections were examined following explantation of the animal's native heart (top) and of the allograft (bottom). By the time of fulminant acute rejection there was pronounced LV hypertrophy. (E) There was also notable scarring and inflammation of the allograft (bottom) at time of explantation noted on gross pathology. (F) On H&E there was significant lymphocytic infiltration and myocardial degeneration; images shown were taken at 10× magnification. (G) Immunofluorescent staining of the allograft tissue for CD3 and CD8 identified cytotoxic T cell infiltration of tissues; images shown were taken at 20× magnification.

Article Snippet: OCT embedded frozen 10 μm thick sections were stained for both CD3ɛ (Invitrogen MA1-90582, mouse, 1:150) to identify the presence of T cells, and CD8α (BioRad MCA6048GA, rabbit, 1:150) to identify the subset of cytotoxic T cells.

Techniques: Functional Assay, Activity Assay, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: A porcine model of acute rejection for cardiac transplantation

doi: 10.3389/fcvm.2025.1549377

Figure Lengend Snippet: Expansion of T cells in the recipient animals’ circulation over time . Blood samples were collected over the course of each animal's observation period (A) Peripheral blood mononuclear cells (PBMC) were isolated (B) and used for immunophenotyping through flow cytometry (C) Overall cell population changes were pooled from Animals A–E and G using uniform manifold approximation and projection (UMAP) analysis (D) Three timepoints were assessed: baseline, 3 weeks post-transplantation, and at the time of cessation of graft activity. Relative to baseline, at the endpoint there was significant expansion of CD4 + (6.6%) and CD8 + (27.8%) T cells. There was also significant expansion of CD25 + T cells (14.9%), CD56 + natural killer cells (14.2%), and CD21 + B cells (3.2%). The changes in specific cell subtypes from baseline to endpoint are also shown as plots with the blue area corresponding to the baseline and the grey area corresponding to the endpoint (E) .

Article Snippet: OCT embedded frozen 10 μm thick sections were stained for both CD3ɛ (Invitrogen MA1-90582, mouse, 1:150) to identify the presence of T cells, and CD8α (BioRad MCA6048GA, rabbit, 1:150) to identify the subset of cytotoxic T cells.

Techniques: Isolation, Flow Cytometry, Transplantation Assay, Activity Assay

Journal: Journal of Animal Science

Article Title: Evaluation of oxidized beta-carotene on sow and piglet immune systems, sow reproductive performance, and piglet growth

doi: 10.1093/jas/skad066

Figure Lengend Snippet: Effects of dietary treatment of maternal nutrition supplemented with oxidized beta-carotene (OxBC) on piglet Kupffer cell phagocytic activity and CD4, CD8 alpha, and CD335 cell populations analyzed via flow cytometry at birth and weaning

Article Snippet: Mouse antipig cluster of differentiation (CD) 335 with a allophycocyanin fluorescent label, mouse antipig CD8 alpha with a phycoerythrin fluorescent label, and mouse antipig CD4 with a fluorescein isothiocyanate fluorescent label (CAT: MCA5972APC, MCA6048PE, MCA6045F, respectively; Bio-Rad Laboratories, Inc., Hercules, CA, US) were added, respectively, at 10 μL per antibody, covered in foil, and allowed to incubate for 15 min at room temperature (roughly 20 °C).

Techniques: Activity Assay, Flow Cytometry, Control