Cfse 5 6 carboxyfluorescein diacetate n succinimidyl ester

BM-derived progenitor cells were obtained from femurs of female C57BL/6 WT, C2gnt1^−/− or Mgat5^−/− mice (6–8 weeks-old). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1 mM HEPES (Life Technologies), 10 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF) (R&D Systems) and antibiotics/antimycotics for 4 days. After in vitro differentiation, cells were incubated with LPS (1 μg/ml; Invitrogen) for 16 h at 37 °C in the presence or absence of rGal-7 (20 μg/ml) or lactose (30 mM; Sigma). The phenotype of MDSCs was determined by flow cytometry as described [45 (link)]. MDSCs (2 × 10⁴ cells) were co-cultured with C57BL/6 splenocytes (2 × 10⁵ cells) in RPMI 1640 supplemented with 10% FBS, 50 μM 2-ME, 1 mM HEPES, anti-CD3 mAb (1 μg/ml), anti-CD28 mAb (1 μg/ml) and antibiotics/antimycotics for 4 days. The ability of MDSCs to control T-cell proliferation was assessed by flow cytometry following incubation with 1 μM 5(6)-carboxyfluoresceindiacetate N-succinimidyl ester (CFSE; eBioscience).

Cryopreserved PBMC were thawed, resuspended in pre-warmed PBS with 0.1% BSA at a final concentration of 10⁶ cells/ml and labelled with 750 nM CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester; Molecular Probes^™) for 10 min at 37°C, 5% CO₂. The staining was quenched by adding 5 volumes of ice-cold R10 followed by a 5-min incubation on ice. The cells were pelleted, washed and plated in 96-well round-bottom plates at a concentration of 1 x 10⁶ cells/well. The CFSE-labelled cells were then stimulated with 1.5 μg/ml of each peptide in personalized pools or 1 μg/ml SEB (positive control) and R10 (negative control) for 5 days, stained with a dead cell marker (LIVE/DEAD Fixable Aqua stain; Invitrogen) and anti-CD4-BV605 (BioLegend), anti-CD3-ECD (Beckman Coulter) and anti-CD8-Aleva Fluor 700 (eBioscience) mAbs, fixed and acquired on a BD LSR II flow cytometer. Data analysis was performed using FlowJo software (Tree Star Inc.) with gating shown in S1 Fig.

To interrogate the capacity of MSCs to suppress T-cell proliferation the cells were co-cultured with peripheral blood mononuclear cells (PB-MNC). 1.5 × 10⁵, 0.75 × 10⁵ or 0.3 × 10⁵ MSCs were suspended in RPMI 1640 medium supplemented with 5 % FBS and penicillin–streptomycin (all from Life Technologies) and plated in 48-well plates. The MSCs were allowed to adhere onto the plates in an incubator before the PB-MNCs were added.
PB-MNCs were isolated from buffy coats by Ficoll-Paque Plus (GE Healthcare, Helsinki, Finland) gradient centrifugation and labelled with 2.5 µM CFSE [5(6)-carboxyfluorescein diacetate N-succinimidyl ester, Molecular Probes] in 0.1 % HSA-PBS (human serum albumin, Sanquin, Espoo, Finland) for 5 min at room temperature. 1.5 × 10⁶ labelled PB-MNCs were then added to the co-culture. For T-cell activation 0.1 µg/ml of CD3 antibody (clone Hit3a, BioLegend, San Diego, CA, USA) was added to the wells. T-cell proliferation was recorded after 4 days of co-culture as dilution of fluorescent dye by flow cytometry. The division index (Flow Jo software v.7.6.1) was used to represent the extent of cell division.

Cryopreserved human PBMCs or isolated mouse splenocytes were resuspended in pre-warmed PBS with 0.1% BSA at a final concentration of 10⁶ cells/ml and labelled with 750 nM CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester; Molecular Probes™) for 10 min at 37°C, 5% CO₂. The staining was quenched by adding 5 volumes of ice-cold R10 followed by a 5-min incubation on ice. The cells were pelleted, washed and plated in 96-well round-bottom plates at a concentration of 1x10⁶ cells/well. The CFSE-labelled cells were then stimulated with 1 μg/peptide/ml of indicated peptide pools, or with 5 μg/ml whole Pfs25 protein, or 1 μg/ml staphylococcal enterotoxin B (SEB) (positive control) and R10 (negative control) for 6 days, stained with a dead cell marker (LIVE/DEAD Fixable Aqua stain; Invitrogen) and anti-CD4⁺-APC (BioLegend), anti-CD3-Alexa-Fluor 700 (BioLegend) and anti-CD8-Alexa Flour 780 (Biolegend) mAbs and acquired on a BD LSR II flow cytometer. Data analysis was performed using FlowJo software (Tree Star Inc.)