Eflour450

BALB/c or C57Bl/6 mDCs were seeded at 3.2 × 10⁵ cells/ml in 24-well plates (Thermo Scientific, Dreieich, Germany) and stimulated with the indicated equimolar concentrations of rFlaA, rArt v 1, rFlaA + rArt v 1, rFlaA:Artv1, or rFlaA:Artv1^hyp for 24 h. 10 µg/ml LPS (#L5886, Sigma Aldrich, Taufkirchen, Germany) served as positive control. Supernatants were analyzed for cytokine secretion by ELISA. The activation of mDCs was assessed by FACS using anti-mouse FITC-conjugated CD40 and phycoerythrin (PE)-conjugated CD69 mAbs (eBioscience, Frankfurt, Germany). Additionally, cells were stained with anti-mouse pacific blue-conjugated CD11b (Invitrogen, Thermo Fisher Scientific), allophycocyanin (APC)-conjugated CD11c (BD Bioscience, Heidelberg, Germany), and PE-Cy5-conjugated B220 (BD Bioscience) with their respective isotype controls. FITC or PE intensity of CD11b⁺CD11c⁺B220⁻ (mDC) cells was quantified by FACS using a LSR II flow cytometer (BD Bioscience, Heidelberg, Germany). For analysis of cell viability mDC were treated as indicated and stained for dead cells using the fixable viability dye eFlour450 (eBiosciences, Frankfurt, Germany). Data were analyzed using FlowJo 10 (Treestar Inc., Ashland, OR, USA).

Mice were sacrificed seven days after immunization and splenocytes were isolated as previously described [6 (link)]. In brief, red blood cells were lysed using ACK buffer and then splenocytes were counted using a Z1 Coulter counter. For tetramer analysis, splenocytes were immediately blocked for Fc (Tonbo Bioscience) and stained for B8R tetramer (AF488, 1:300, NIH Tetramer Facility, Atlanta, GA) followed by staining with anti-CD3 (PeCy7, 1:100, clone 145-2C11) and anti-CD8α (eFlour450, 1:200, clone 145-2C11). Cells were then stored overnight at 4C in a 1:1 of IC Fixation Buffer (ThermoFisher Scientific) and FACS buffer. For analysis of cytokine production, 1.7x10⁶ cells were plated in a 96 well dish and incubated for five hours in the presence of B8R_20-27 (TSYKFESV) or OVA_257-264 (SIINFEKL) peptides and brefeldin A (eBioscience). Splenocytes were then subjected to FC block (Tonbo Bioscience) and stained with anti-CD3 (FITC, 1:200, clone 145-2C11) and anti-CD8α (eFlour450, 1:200, clone 53–6.7) before treatment with fixing and permeabilization buffers (eBioscience). Cells were then further stained with anti-IFNγ (APC, 1:300, clone XMG1.2). Samples were acquired using the LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR).

Antibodies used were anti-CD4 (RM4-5) conjugated to PerCpCy5.5, anti-CD3 (145-2C11) conjugated to FITC or anti-CD3 (500 A2) V500, anti-CD44 (1M7) conjugated to AlexaFlour700 (all BD Biosciences), anti-CD11c (N418), anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-F4/80 (BM8) conjugated to eFlour450 and anti-FoxP3 (FJK-16S) conjugated to APC (all eBiosciences), anti-CD8a (5H10) conjugated to Pacific Orange (Invitrogen) or anti-CD8a (53-6.7) conjugated to PE-Cy7, anti-CD62L (MEL-14) conjugated to APC-Cy7 (BD Biosciences). FoxP3 was stained intracellularly using FoxP3/Transcription Factor Fixation/Permeabilization kit (eBiosciences). Data were collected on an LSR Fortessa flow-cytometer (BD) and analyzed using FlowJo (Treestar) software.

Surface levels of CD4, BST-2, CCR7 and NTB-A were assessed by staining cells with their appropriate antibodies at 4°C for 30 min in buffer (1 × PBS + 3% FBS). An additional step including a secondary antibody was necessary to detect BST-2 surface levels. A viability dye, eFlour 450 (eBioscience) was then used to distinguish live from dead cells. Fixation was achieved using 0.5% Paraformaldehyde (PFA).
In experiments involving surface analysis of CD4 and detection of intracellular p24, cells were first probed with anti-APC-CD4, stained with eFluor 450, permeabilized (Cytofix/Cytoperm: BD Biosciences) and then stained with mouse-anti-FITC-p24. Total levels of CD4 in primary CD4⁺ T cells were measured by staining cells with eFlour 450, permeabilization and then probing with anti-APC-CD4. All data was collected on a BD FACS CantoII and analyzed with FlowJo software.

A single-cell suspension of splenocytes from naïve syngeneic mice was diluted to 1.5 × 10⁸/ml in RPMI1640 containing 10% FBS and 2% penicillin and streptomycin pulsed at 37°C with or without 5 μg/ml peptides as described previously [19 (link)]. After 4 h, eflour450 (eBioscience, 65-0842-85) at 5 mM (high concentration) was used to label peptide-pulsed cells at room temperature in the dark. Non-peptide-pulsed cells were labelled with a low concentration of eflour450 at 0.5 mM. After being rinsed three times with PBS, 4 × 10⁶ labelled and peptide-pulsed cells and an equal number of labelled non-peptide-pulsed cells were adoptively transferred by tail vein injections into mice that had previously been immunized. Six hours later, the percentage of labelled cells in spleens was detected with LSRFortessa flow cytometry (BD) and analyzed by FlowJo (TreeStar). The following formula calculated the specific cell lysis: Specific cell lysis ability% = (1-(percentage of cells incubated with peptide/percentage of cells incubated without peptide)) x100%.

To analyze the migratory capacity of circulating human ILC2s under defined experimental conditions, freshly isolated CRTH2⁺ human blood cells were labeled with the hematopoietic lineage cocktail (eFlour450, eBioscience) and fluorescent antibodies targeting CD11c (VioBlue, MJ4-27G12, Miltenyi Biotec), CD161 (FITC, 191B8, Miltenyi Biotec) and CRTH2 (PE, BM16, Miltenyi Biotec). 1.6 × 10⁵ stained CRTH2⁺ cells were suspended in 80 μl X-Vivo 15 medium (Lonza, 1% P/S) and applied to the upper insert of a 96 well-plate with 3 μm pore size (Corning). If indicated, a CCR6 blocking antibody (50 μg/ml in the insert, MAB195, R&D) or the respective isotype control antibody (BioLegend) were added. The bottom well was filled with 235 μl X-Vivo 15 medium and rh CCL20 (10 ng/ml and 100 ng/ml, Immunotools) as indicated. CCL25 (100 ng/ml, Immunotools) served as negative control, PGD2 (10 nM, Merck) as positive control (31 (link)). After 4 h at 37°C the number of migrated ILC2s in the bottom chamber was determined as Lin^negCD161⁺CRTH2⁺ lymphoid cells via flow cytometry. The migration index was defined as relative migration of Lin^negCD161⁺CRTH2⁺ cells attracted toward a chemotactic stimulus compared to those attracted by medium alone.