The ability to phenotype mixed cell populations and to isolate specific cell types for controlled analysis has broad application in biological research. This breadth of utility makes cytometers and cell sorters ideally suited for placement in a shared resource setting. This month we introduce the Unified Flow Core (UFC), the primary flow cytometry and sorting resource at the University of Pittsburgh.
Unified Flow Core
UFC currently supports 11 cell analyzers and four cell sorters 
at the Biomedical Science Tower (BST) and BST South in Oakland and The Assembly in Shadyside.
Instrument options include:
- traditional fluorescent analyzers and sorters that analyze or sort on up to 16 fluorescent channels;
- traditional sorter in BSL2+ containment, available at the BST location;
- spectral analyzers and sorters that analyze or sort on 64 fluorescent channels;
- imaging analyzer that can analyze with up to 12 fluorescent channels while simultaneously imaging cell morphology and
- consultation with UFC staff for expert assistance on panel selection and design to make optimal use of instrument capabilities.
To access UFC, complete the new user form and notify core staff at flowcore@pitt.edu. A staff member will contact you to schedule training. A complete description of training options and procedures for scheduling equipment is available on the policy section of the UFC website. Once training is complete, users can operate analyzers independently. Due to their more complex nature and greater potential for experimental failure, sorters must be operated by UFC staff.
Testimonials
Diana Metes (Departments of Surgery and of Immunology) analyzed peripheral blood mononuclear cells collected from kidney transplant patients at multiple time points from pre- to 24 months post-transplant using a panel of 43 intra- and extracellular markers and UFC’s Aurora spectral flow cytometer. This analysis revealed expanding populations of circulating follicular helper T cells (cTFH) and activated B cells in patients developing donor-specific antibodies, a first step in antibody-mediated rejection. With a subset of markers specific to these populations, Metes’s group used the FACSAria II cytometer to isolate them for controlled co-culture and testing. This study opened new avenues of investigation that could lead to improved clinical outcomes for kidney transplant recipients.¹
Hassane Zarour (Departments of Medicine, of Immunology, and of Dermatology) ran a clinical trial investigating the safety and efficacy of responder-derived fecal microbiome transplant (FMT) to improve response rates in patients with advanced melanoma recalcitrant to anti-PD1 therapy. Using five staining panels of 29 intra- and extracellular markers each, and the UFC Aurora flow cytometer, the Zarour team characterized the immune cell profile of subjects at three time points (days 0, 21 and 42 of combined anti-PD1 and FMT treatment). To investigate intratumoral immune response, UFC resources were also used to sort CD45+ cells for single cell transcriptomic analysis. While larger clinical studies are required to draw any definitive conclusions, these results suggest that FMT can modulate anti-PD1 recalcitrance in some cases of advanced melanoma through increased activation and differentiation of CD8+ T cells.²
Fadi Lakkis (Departments of Surgery, of Immunology, and of Medicine) used imaging flow cytometry to investigate antigen presentation pathways in allograft rejection. Using transplant models in mice, his group illustrated that host dendritic cells coated with donor major histochemical complex-peptide complexes within the transplanted organ are a major component of the immune response leading to allograft rejection. This new understanding of the drivers of rejection opens new possibilities for treatments to interrupt transplant rejection.³
Mark Shlomchik (Department of Immunology) employed the core’s cell sorters to study subsets of memory B cells (see figure.) Using a powerful mouse model for tracking B cell memory, the lab identified six distinct subsets of memory T cells, which, after sorting, were studied by RNA sequencing, chromatin profiling and restimulation. These analyses revealed that the different subsets of B cells develop via unique developmental pathways. These studies have the potential to inform better approaches to track and induce B cell memory by vaccines and other interventions.⁴
1. Louis K, Macedo C, Bailly E, Lau L, Ramaswami B, Marrari M, Landsittel D, Chang A, Chandran U, Fadakar P, Yamada M, Chalasani G, Randhawa P, Zeevi A, Singh H, Lefaucheur C, Metes D. Coordinated Circulating T Follicular Helper and Activated B Cell Responses Underlie the Onset of Antibody-Mediated Rejection in Kidney Transplantation. J Am Soc Nephrol. 2020 Oct;31(10):2457-2474. doi: 10.1681/ASN.2020030320. Epub 2020 Jul 28. PMID: 32723838; PMCID: PMC7608995.
2. Davar D, Dzutsev AK, McCulloch JA, Rodrigues RR, Chauvin JM, Morrison RM, Deblasio RN, Menna C, Ding Q, Pagliano O, Zidi B, Zhang S, Badger JH, Vetizou M, Cole AM, Fernandes MR, Prescott S, Costa RGF, Balaji AK, Morgun A, Vujkovic-Cvijin I, Wang H, Borhani AA, Schwartz MB, Dubner HM, Ernst SJ, Rose A, Najjar YG, Belkaid Y, Kirkwood JM, Trinchieri G, Zarour HM. Fecal microbiota transplant overcomes resistance to anti-PD-1 therapy in melanoma patients. Science. 2021 Feb 5;371(6529):595-602. doi: 10.1126/science.abf3363. PMID: 33542131; PMCID: PMC8097968.
3. Hughes AD, Zhao D, Dai H, Abou-Daya KI, Tieu R, Rammal R, Williams AL, Landsittel DP, Shlomchik WD, Morelli AE, Oberbarnscheidt MH, Lakkis FG. Cross-dressed dendritic cells sustain effector T cell responses in islet and kidney allografts. J Clin Invest. 2020 Jan 2;130(1):287-294. doi: 10.1172/JCI125773. PMID: 31763998; PMCID: PMC6934226.
4. Callahan D, Smita S, Joachim S, Hoehn K, Kleinstein S, Weisel F, Chikina M, Shlomchik M.
Memory B cell subsets have divergent developmental origins that are coupled to distinct imprinted epigenetic states. Nat Immunol. 2024 Mar;25(3):562-575. doi: 10.1038/s41590-023-01721-9. Epub 2024 Jan 10. PMID: 38200277.