Fortessa

For live‐cell luminescence microscopy, cells were cultured in N2B27+2iLIF for at least two passages before 30,000 cells were seeded on E‐Cadherin in fluorodishes (WPI, Cat#FD35‐100) and incubated in N2B27+2iLIF overnight. The next day, the medium was changed to N2B27 supplemented with 1 mM luciferin and 0.5 μl of RealTime Glo Cell Viability Assay Substrate, and image acquisition was started.
For differentiation assays after cell sorting, cells were seeded at a density of 60,000 cells/well of a 6‐well plate coated with gelatin. Two days later, the medium was exchanged for fresh N2B27 and after 4 days differentiation outcomes were assessed by flow cytometry on a BD Fortessa.
To direct differentiation towards the mesendoderm, cells were seeded at a density of 60,000 cells/well of a gelatin‐coated 12‐well plate in N2B27 medium supplemented with 3 μM of CHIR99021. Three days later, differentiation outcomes were assessed by flow cytometry on a BD Fortessa. To direct differentiation towards the neuroectoderm, cells were seeded at a density of 60,000 cells/well of a gelatin‐coated 6‐well plate or 30,000 cells/well of a gelatin‐coated 12‐well plate in N2B27 medium supplemented with 1 μM SB‐431542 and 25 ng/ml of bFGF. Four days later, differentiation outcomes were assessed by flow cytometry on a BD Fortessa.

Directly upon arrival from Guinea-Bissau all whole blood samples, collected in Cyto-Chex BCT tubes (Streck), were processed and analyzed on a BD Fortessa instrument (BD Biosciences) as previously described (22 (link)). Likewise, it was confirmed that the expression patterns of markers were equivalent between Cyto-Chex-stabilized blood and fresh blood samples (data not shown). In brief, the blood samples were incubated in Lysis buffer (BD Biosciences) at a ratio of 1:6 for 5 min before centrifugation and washing in PBS/FCS (2%). After treatment with DNase (6 U/ml), the cells were extracellularly stained in PBS/EDTA (2mM) for 10 min at 37°C and further 20 min at room temperature. Permeabilization was performed with the FoxP3 kit (eBiosciences) prior to 60 min intracellular staining. Antibodies used for staining of extracellular (2B4, CCR7, CD3, CD4, CD8, CD14, CD19, CD38, CD45RO, CD226, CXCR5, HLA-DR, PD-1 and TIGIT) and intracellular (Eomes) antigens, clones, fluorophores and suppliers are listed in Supplementary Table S1. The cells were resuspended in Cytofix Buffer (BD Biosciences) and analyzed on a BD Fortessa.

The cultured TIL phenotypes after culture were characterized using flow cytometry with anti-CD3 (Cat#: 555339, 1.5 μL/10⁶ cells), anti-CD4 (Cat#: 557871, 2 μL/10⁶ cells), anti-CD8 (Cat#: 563823, 2 μL/10⁶ cells), anti-CD56 (Cat#: 56275, 3 μL/10⁶ cells), and anti-PD1 (Cat#: 561272, 5 μL/10⁶ cells) for 30 minutes on ice in the dark [35 (link), 37 (link)]. Thereafter, the cells were washed once with PBS and resuspended in 400 μL PBS. 7AAD was used to distinguish live cells and dead cells, and the cells were run on a BD Fortessa (BD Biosciences). Fluorescence minus one (FMO) was used as the negative control. Moreover, FlowJo software was used to analyze the data generated by flow cytometry. FoxP3 staining was conducted using an intracellular staining protocol from BD Biosciences as follows: anti-CD3 and anti-CD4 were stained for 30 minutes on ice in the dark; TILs were washed, fixed, and permeabilized following protocols for BD Fix Buffer I (Cat#: 557870, BD Biosciences, USA) and Perm Buffer III (Cat#: 558050, BD Biosciences, USA). The cells were washed thrice with Perm Buffer III and incubated with anti-FoxP3 (Cat#: 560460, 5 μL/10⁶ cells) for 30 minutes on ice in the dark. The cells were run on a BD Fortessa (BD Biosciences). Fluorescence minus one (FMO) was used as the negative control. FlowJo software was used to analyze the data generated by flow cytometry.