Facs buffer

Digested tumor or adjacent normal cells were centrifuged and resuspended at 5 × 10⁵–2 × 10⁶ cells/100 µL FACs buffer (Miltenyi, Bergisch Gladbach, Germany). Cells were divided into no antibody control, viability analysis panel (Propidium iodide, Hoeschst33342, DRAQ7), IgG control (REA-VioBlue, REA-FITC, REA-PE, REA-APC, REA-PE-Vio770), staining panel 1 (1:50 CD31-VioBlue (Miltenyi 130-117-227), 1:11 CA9-PE (Miltenyi 130-110-057), 1:10 PDGFRβ-APC (Miltenyi 130-105-322), 1:10 PDGFRα-APC (Miltenyi 130-115-239), 1:50 CD326-PE-Vio770 (Miltenyi 130-111-002), 1:50 CD45-FITC (Miltenyi 130-110-631), Propidium Iodide), or staining panel 2 (1:50 CD10-VioBlue (Miltenyi 130-114-509), 1:11 CA9-PE, 1:50 CD105-PE-Vio770 (Miltenyi 130-112-167), CD184-APC (Miltenyi 130-098-357), 1:50 CD326-PE-Vio770, Propidium Iodide), and incubated in the dark at 4 °C for 15 min. Cells were washed two times with flow buffer and analyzed using Miltenyi AutoMACs flow cytometer. Side and forward scatter gating were determined using viability analysis panel to identify cells vs. debris. Doublets were excluded using SSR-H versus SSR-L. Positive gating for each marker was determined using IgG control. Compensation was performed using the MACS Comp Bead kit, anti-REA (Miltenyi 130-104-693).

Quantitative flow cytometry was performed using the MACSQuant^® VYB Flow Cytometer (Miltenyi Biotech, Bergisch Gladbach, Germany). Cell pellets were resuspended in FACS buffer (Miltenyi Biotec). To sort out apoptotic cells, samples were stained 1:1000 with Aqua Fluorescent Reactive Dye (Thermo Fisher). The emission of ZsGreen was measured to determine the efficiency of the DD-Dre system, while the emission of mCherry served as a transfection control. The following gating strategy was used: forward scatter-area (FSC-A) vs. side scatter-area (SSC-A) dot blots for cell identification. Single cells were gated via side scatter-height (SSC-H) vs. SSC-A and forward scatter-height (FSC-H) vs. FSC-A dot blots. Dead and apoptotic cells are gated out according to their Aqua fluorescence. Data were analyzed by MACSQuant^® Analyzer 10 (Miltenyi Biotech) and Flowjo (Treestar, Woodburn, OR, USA).

Following the immunization, the magnitude of differentiation of splenic T lymphocytes was evaluated by using FACS analysis. At week 1 post immunization, splenocytes were aseptically isolated from five mice of each group. The prepared splenocytes were washed in PBS and RPMI1640 medium, and seeded in 96 well cell culture plate at the density of 5 × 10⁵ cells per well. After washing with FACS buffer (MiltenyiBiotec, Bergisch Gladbach, Germany), the cells were stained with anti-CD3-PE, CD4-perCP-vio700 and CD8-FITC antibodies (MiltenyiBiotec). The percentage of CD4⁺ or CD8⁺ population was estimated in the CD3⁺ gated cells.

Following vaccination, the magnitude of the differentiation of splenic T cells was assessed using fluorescence-activated cell sorting (FACS), as previously described. Briefly, the mice were euthanized, and spleens were aseptically isolated at day 10 post-immunization. The prepared splenocytes (1 × 10⁶ cells/mL) were incubated with a mixture of fluorescent-labeled antibodies, such as anti-mouse CD3a-PE, anti-mouse CD4-perCP-vio700, and anti-mouse CD8a-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 4 °C in the dark. The samples were washed twice with FACS buffer (Miltenyi Biotec) and resuspended in 200 μL of FACS buffer. The cells were then examined with the MACSQuant^® Analyzer (Miltenyi Biotec). The results were further analyzed using FlowJo Software (Tree Star Inc., CA, USA).

An alteration in T cell subsets of the immunized mice was assessed using fluorescence-activated cell sorting (FACS) (Jawale and Lee, 2014 (link)). Spleen cells were isolated from the immunized and non-immunized mice at day 7 PI and 1 × 10⁶ of the cells were seeded into a 96-well cell culture plate. The splenic cells were stained with the surface markers containing anti-mouse CD3a-PE and anti-mouse CD4-perCP-vio700 (Miltenyi Biotec, Bergisch Gladbach, Germany). After the stained cells were washed twice in FACS buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), the stained CD3+ and CD3+CD4+ splenic cells were sorted with an MACSQuant^® Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). The altered FACS profiles of CD3+ and CD3+CD4+ splenic T cell subsets in the immunized mice were analyzed in comparison to those from non-immunized mice.