Jolly Technologies ID Flow 3.4 P
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Immunoplatelet counting. (a) Illustration of the main optical elements of a flow cytometer. Cells in suspension flow in a single-file are illuminated in the flow cell where they scatter light (forward scatter: signal depends mainly on cell size, and side scatter: signal depends on complexity and granularity) and emit fluorescence. (b) Typical biparametric dot plot showing platelets, identified by their low forward scatter, compared to red cells, and their staining by CD41 and CD61.
Optical platelet counting. (a) Illustration of the main optical elements. Cells in suspension flow in a single-file, and when illuminated in the flow cell, they scatter light (forward scatter: the signal depends mainly on cell size, and side scatter: the signal depends on the complexity and granularity). (b) Typical biparametric dot plot showing platelets in turquoise and debris in white.
Fluorescence platelet counting. (a) Illustration of the main optical elements. Cells in suspension flow in a single-file, and when illuminated in the flow cell where they scatter light (forward scatter: the signal depends mainly on cell size, and side scatter: the signal depends on the complexity and granularity) and fluorescence. (b) Typical biparametric dot plot showing platelets in turquoise, immature platelets in green, and RBCs in blue.
The extent of this inhibition was monitored by flow cytometry of CD11c-positive DCs infected with GFP-expressing Brucella or Salmonella, for surface expression of classical maturation markers (eg CD80, CD86 and MHC molecules). Analysis of the median of fluorescence (Figure 4A) or the geometric mean (data not shown) in the CD11c+GFP+ populations showed a consistently lower surface expression of all co-stimulatory and MHC class II molecules in cells infected with B. abortus than in Salmonella-infected cells (Figure 4A and 4B). However, statistical difference between B. abortus and Salmonella was only observed for CD86 expression (p = 0.011). This is probably due to the fact that in the case of Brucella-infected DCs two populations were observed (overlay histograms, Figure 4A and 4B), one with a moderately increased surface levels in comparison to control cells and the other with similar levels of expression to Salmonella-activated DCs. This is in agreement with microscopy observations where a high proportion of MHC class II molecules remained intracellular in Brucella-infected cells (Figure 2A). Surprisingly, we did not detect any significant difference in the surface expression of MHC class I molecules in cells infected by the two pathogens (Figure 4B), thus suggesting the expression of MHC class I molecules is controlled differently than MHC class II and co-stimulatory molecules. Together, these data confirm that maturation of Brucella-infected DCs is impaired.
Intracellular IL-12 (p40/p70) expression was further analysed by flow cytometry. DCs incubated with either media alone or infected with GFP-expressing Brucella or Salmonella were labelled at 4 and 24 h post-inoculation with anti-CD11c and IL-12 (p40/p70) antibodies. Analysis of IL-12 expression was carried out on CD11c+ cells (Figure 5B). IL-12 expression in Salmonella-infected DCs peaked after 4 h, similarly to what observed with E. coli LPS (data not shown), whereas in Brucella-infected DCs, IL-12 expression was only detected after 24 h (Figure 5B). Similar results were obtained when analysing GFP-positive populations for each pathogen (data not shown).
The primary antibodies used for immunofluorescence microscopy were: cow anti-B. abortus polyclonal antibody; hamster anti-CD11c (N418; Biolegend); affinity purified rabbit Rivoli antibody against murine I-A [20]; rat anti-mouse LAMP1 ID4B (Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, University of Iowa); mouse antibody FK2 (Biomol); mouse anti-KDEL (Stressgen). Monoclonal anti-calnexin antibody was kindly provided by Dr. D. Williams (University of Toronto). For flow cytometry allophycocyanin conjugated-anti-CD11c antibody (HL3) was used in all experiments along with either phycoerythrin-conjugated CD40, CD80, CD86, IA-IE (MHC class II) or H2-2Kb (MHC class I) all from Pharmingen. Appropriate isotype antibodies were used as controls (data not shown). For intracellular labelling of IL12 the phycoerythrin-conjugated IL-12 (p40/p70) monoclonal from Pharmingen was used.
For flow cytometry, infected DCs were collected and stained immediately before fixation. Isotype controls were included as well as DCs infected with non-gfp B. abortus as control for autofluorescence. Cells were always gated on CD11c for analysis and at least 100,000 events were collected to obtained a minimum of 10,000 CD11c-positive and GFP-positive events for analysis. A FACScalibur cytometer (Becton Dickinson) was used and data was analysed using FlowJo software (Tree Star).
Measurement of lactate dehydrogenase (LDH) release in the supernatant of cells infected with different strains was carried out using the Detection Kit (Roche) as indicated by the manufacturer. The percentage of cytotoxicity corresponds to the ratio between the experimental value subtracted by the negative control (spontaneous LDH release) and the maximum LDH release (triton lysed cells) subtracted by the negative control. For detection of 7-AAD, cells were infected with GFP-expressing strains as described above and collected at 24 h in cold PBS. Cells were then labelled first for CD11c-APC, washed several times in cold PBS and then incubated with 7-AAD following the manufacturer's instructions (BD Pharmingen). The flow cytometric analysis were performed on fixed cells within 20 min.
We would especially like to acknowledge Chantal de Chastellier for advice with electron microscopy; Cristel Archambaud for flow cytometry on infected intestinal samples; Sandrine Henri for help with the antigen presentation assays; Markus Schnare for the TLR9 construct; David Williams for the anti-calnexin antibody; Shizuo Akira for the TLR2, TLR4, MyD88, and TLR4 deficient mice; and Bruce Beutler for the Lps2 (TRIF) deficient mice. We would also like to acknowledge Beatrice de Bovis and the CIML histology core facility. We thank the PICsL imaging and FACS core facility for expert technical assistance. We are also very grateful to Stephane Meresse for helpful discussions and critical reading of the manuscript.
We would like to thank the following people for their assistance with this manuscript: Tilak Prasad and Surabhi S.V. for helping with flow cytometry, Anurup K.G. for technical support with confocal imaging, and T. R. Santhosh Kumar for helping with lab consumables and technical support.
Fetal liver cells were stained for CD41 as described above and fixed with 5% formalin for 15 min. Cells were permeabilized in PBS containing 0.25% Tx-100 for 5 min at 4C. DNA was stained with DAPI for 20 min and DNA content in CD41+ cells was determined by flow cytometry.
Washed platelets were stimulated or not with ADP (25 µM) or collagen-related peptide (CRP) (1 µg/ml),under non-stirring conditions. After 15 minutes of activation, saturating concentrations of FITC-conjugated CD62 anti-P-selectin and PE-conjugated JON/A antibodies were added to the platelets, and incubations were continued for additional 15 minutes in the dark. Samples were fixed before the analysis with a FACS Calibur flow cytometer (BD Biosciences).
We thank Y. Maréchal for discussion and ideas, Laoura Sacré and Aurélie Fastré for technical help, A. Scoumanne for comments on the final manuscript, the GIGA technology plate-forms (GIGA-Research Centre, Université de Liège) for help with imaging, flow cytometry, immunohistochemistry and animal husbandry, and P. J. Cullen and O. Leo for specific reagents. 153554b96e
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