PE Mouse Anti-Human CD27

Protocol tips

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	For the ex vivo immunophenotyping experiments, frozen samples were used and were thawed in R10 supplemented with 20 µg/ml DNase I from bovine pancreas (Sigma-Aldrich). After extensive washing with PBS (Sigma-Aldrich), the cells were stained immediately with the Zombie Aqua Fixable Viability kit (BioLegend) for 15 min at room temperature. Then, the cells were washed and stained with the combination of mAbs for 30 min at 4°C.	All data were acquired on a FACS Symphony A5 flow cytometer (BD Biosciences) equipped with five lasers (UV, 350 nm; violet, 405 nm; blue, 488; yellow/green, 561 nm; red, 640 nm; all tuned at 100 mW, except UV tuned at 60 mW) and capable to detect 30 parameters. Flow cytometry data were compensated in FlowJo by using single stained controls (BD Compbeads incubated with fluorescently conjugated antibodies)

Protocol tips
For the ex vivo immunophenotyping experiments, frozen samples were used and were thawed in R10 supplemented with 20 µg/ml DNase I from bovine pancreas (Sigma-Aldrich). After extensive washing with PBS (Sigma-Aldrich), the cells were stained immediately with the Zombie Aqua Fixable Viability kit (BioLegend) for 15 min at room temperature. Then, the cells were washed and stained with the combination of mAbs for 30 min at 4°C.
Downstream tips
All data were acquired on a FACS Symphony A5 flow cytometer (BD Biosciences) equipped with five lasers (UV, 350 nm; violet, 405 nm; blue, 488; yellow/green, 561 nm; red, 640 nm; all tuned at 100 mW, except UV tuned at 60 mW) and capable to detect 30 parameters. Flow cytometry data were compensated in FlowJo by using single stained controls (BD Compbeads incubated with fluorescently conjugated antibodies)

Publication protocol

"For the ex vivo immunophenotyping experiments, frozen samples were used and were thawed in R10 supplemented with 20 µg/ml DNase I from bovine pancreas (Sigma-Aldrich). After extensive washing with PBS (Sigma-Aldrich), the cells were stained immediately with the Zombie Aqua Fixable Viability kit (BioLegend) for 15 min at room temperature. Then, the cells were washed and stained with the combination of mAbs purchased from either BD Biosciences, BioLegend, or eBioscience, as listed in Table S1. mAbs were previously titrated to define the optimal concentration, as described (Lugli et al., 2013b). Chemokine receptors were stained for 20 min at 37°C, while all other surface markers (except CD3) were stained for 20 min at room temperature. Intracellular detection of CD3, Ki-67, and transcription factors was performed following fixation of cells with the FoxP3/transcription factor staining buffer set (eBioscience) according to manufacturer’s instructions and by incubating with specific mAbs for 30 min at 4°C.

In parallel, all samples were stained with a panel of mAbs directed to reveal the major immune populations, such as T cells (CD3+), B cells (CD19+), myeloid (CD11b+), and NK subsets (CD16, CD56, and NKp46). Additional panels used for further characterizing the CXCR5+ CD8+ T cell subset are listed in Table S1. Stainings were performed as described above, except for intracellular staining of TCF-1, for which cells were fixed with Transcription Factor Buffer set (BD Biosciences) according to the manufacturer’s instructions. For detection of cytokines, cells were fixed with BD Cytofix/Cytoperm Fixation/Permeabilization Solution kit (BD Biosciences) according to the manufacturer’s instructions. CD107a-specific antibody was added in the culture medium during the stimulation.

All data were acquired on a FACS Symphony A5 flow cytometer (BD Biosciences) equipped with five lasers (UV, 350 nm; violet, 405 nm; blue, 488; yellow/green, 561 nm; red, 640 nm; all tuned at 100 mW, except UV tuned at 60 mW) and capable to detect 30 parameters. Flow cytometry data were compensated in FlowJo by using single stained controls (BD Compbeads incubated with fluorescently conjugated antibodies), as described (Lugli et al., 2017)."

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