Cytofix cytoperm fixation permeabilization kit

T cell co-cultures with either infected BMDCs [see section In vitro T Cell Assays (T Cell Lines)] or from ex vivo peptide-stimulated splenocytes [see section Ex vivo T Cell Analysis (Splenocytes)] were transferred into 96-well V-bottom plates. Cells were incubated with blocking buffer [PBS having 1% bovine serum albumin (BSA)] containing 1 μg/ml ethidium monoazide bromide (Life technologies GmbH, Germany) for 20 min on ice under light exposure for live/dead discrimination. Afterwards, ICS was performed using BD Cytofix/Cytoperm fixation/permeabilization kit according to the manufacturer's protocol (BD Pharmingen, Germany). Briefly, cells were washed twice with blocking buffer and surface stained with anti-CD8-PB or anti-CD4-PB for 30 min on ice. In addition, cells were stained for CD62L (L-selectin) in order to discriminate between T cells that were specifically reactivated after ex vivo antigen restimulation (CD62L negative) and those that were not reactivated in an antigen-specific manner (CD62L positive). Cells were washed and permeabilized with Cytofix/Cytoperm solution for 15 min on ice. Thereafter, cells were washed, incubated with anti-IFNγ, anti-TNFα or anti-IL-2 for 30 min, washed again, fixed with 2% paraformaldehyde (PFA) and subjected to flow cytometry [BD FACSCanto II (BD Bio Sciences, Germany)].

Four hours after electroporation (healthy volunteer donor samples) or two hours after thawing (patient samples), DCs were stained with LIVE/DEAD® Fixable Red Dead Cell Stain (cat# L23102, Thermo Fisher Scientific) prior to fixation and permeabilization for intracellular staining using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (cat# 554714, BD Biosciences) according to the manufacturer's instructions. Subsequently, samples were incubated with mouse anti-RHAMM monoclonal antibody (mAb; clone 2D6, cat# MO20030, Neuromics) or isotype control mouse IgG1 mAb (cat# 349040, BD Biosciences) followed by PE-labeled rat anti-mouse IgG1 mAb (clone X56, cat# 340270, BD Biosciences). RHAMM expression in viable (LIVE/DEAD⁻) cells was determined on a FACScan flow cytometer (BD Biosciences).

Splenocytes from immunized mice were stimulated in vitro with peptides (10 μg/ml) in the presence of GolgiStop and GolgiPlug (BD-Biosciences) and 4 h later they were harvested and stained with antibodies CD3ϵ-Percp-Cy5 (145-2C11), CD4-FITC (RM4-5), CD8-BV421 (53-6.7) from BioLegend (San Diego, CA). Next, cells were fixed and permeabilized using BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit for intracellular staining with IFNγ-PE (XMG1.2). Dead cells were excluded from the analysis using Maleimide (PromoCell; Heidelberg, Germany). Samples were acquired with a Cytoflex (Beckman Coulter; Indianapolis, IN) flow cytometer. All data were analysed using FlowJo software (FlowJo LLC; Ashland, OR).

THP-1 cells, 1 × 10⁶ cells per ml, were co-cultured with ABAs corresponding to final aluminium concentrations ranging from 25 to 100 μg/ml during 1 to 16 h (overnight) at 37 °C. Controls were cells cultured in R10 in the absence of aluminium adjuvant. The cells were harvested, washed with PBS and divided into two fractions. One cell fraction was re-suspended in PBS containing 0.1% (w/v) BSA and 0.1% (w/v) human IgG and surface stained using APC-labelled anti-human HMGB1 or an APC-labelled isotype control. The second fraction was permeabilised using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Bioscience, San Jose, CA, USA) and intracellularly stained using APC-labelled anti-human HMGB1 or an APC-labelled isotype control.
Finally, the cells were analysed by flow cytometry using an Accuri C6 flow cytometer with standard settings.

CD8⁺ T cells were sorted and cultured as described in Sections 2.2 and 2.3. For the intracellular measurement of interferon‐γ (IFN‐γ), granzyme B (GZMB) and perforin‐1 (PRF‐1), the CD8⁺ T cell subsets were incubated with 3 μg/ml brefeldin A (Thermo Fisher Scientific, Waltham, MA) for 6 h. The harvested cells were then stained with anti‐CD8‐V450 (for details see above), anti‐IFN‐γ‐Alexa Fluor 488 (clone: B27, RRID:AB_396827), anti‐granzyme B‐Alexa Fluor 647 (clone: GB11, RRID:AB_10897997) and anti‐perforin‐1‐PE‐CF594 (clone: δG9 RRID:AB_2738410), all obtained from BD Biosciences (San Diego, CA) using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Biosciences San Diego, CA) according to the manufacturer's instructions. All antibodies were titrated to determine the optimal concentration for use. The stained samples were acquired with a FACSymphony™ A5 Cell Analyzer (BD Biosciences, Heidelberg, Germany). The FMO stainings were used for gating. The data were analysed using FlowJo software V10.7.1 (BD Life Sciences).