Enunciados científicos: las hipótesis y su contrastación

Single cell suspensions were stained with fluorescein isothiocyanate (FITC)-, R- phycoerythrin (PE)-, PE-cyanine 5 (PE-Cy5)-, or allophycocyanin (APC)-conjugated antibodies, and detected by a FACS Calibur flow cytometer (BD Biosciences) or sorted using a FACSAria I (BD Biosciences), as indicated. Nonviable cells were excluded from analyses by propidium iodide (PI; Sigma-Aldrich) or 7-Aminoactinomycin D (7-AAD; BioLegend) staining, and appropriate gating. The data were analyzed using CellQuest (BD Biosciences) software.

When mouse PB was analyzed by flow cytometry, the samples were treated to lyse red blood cells before initiating the staining protocol. PB samples collected in anticoagulant were transferred to 15 ml conical tubes and centrifuged at 300 ×g for 5 min at 4°C. After discarding the supernatant, the pellet was resuspended in 5 ml of red blood cell (RBC) lysis solution (composition in Table 2.5), and incubated for 5 min at RT. Then, the samples were centrifuged at 300 ×g for 5 min at 4°C, the supernatant was discarded, and the pellets were resuspended in cold PBS.

2.4.1. Immunostaining for detection of T-cell markers and RANKL

A volume of single cell suspension containing 1×106 _{cells, was added to each 5 ml round-} bottom polystyrene tube (BD Falcon) plus 1 ml of ice-cold FACS solution (composition in Table 2.5). After centrifugation at 300 ×g for 5 minutes at 4ºC, the cells were stained with fluorochrome-labeled antibodies alone or in combination at the indicated dilutions (Table 2.1) in 50 µl of FACS solution, and incubated for 1 hour on ice, in the dark. Then, the cells were washed twice with 1 ml of cold FACS solution and resuspended in 1 ml of 10 mM NaN3 in PBSsolution, and analyzed. Isotype negative controls and cells stained with each antibody individually were also prepared to adjust the FACS instrument settings

2.4.2. Detection of intracellular lymphotoxin-β (LTβ)

Intracellular lymphotoxin-β was detected using a hamster anti-mouse LTβ (BBF6.BF12) monoclonal antibody (Browning et al., 1997). This antibody and the negative control, anti- KLH armenian hamster IgG Ha4/8, were provided by Jeff Browning (Boston University School of Medicine, Boston, USA). Both primary antibodies were visualized using FITC- conjugated goat anti-hamster (Armenian) IgG (BioLegend) secondary antibody. More specifically, a volume of suspension containing 0.5×106 _{cells was transferred to each 5 ml} round-bottom polystyrene tube (BD Falcon) and washed with 1 ml of ice-cold PBS. For washing, cells were centrifuged at 300 ×g for 5 minutes at 4ºC and the supernatant was discarded. Then, cells were fixed with 200 µl of 0.5% paraformaldehyde (PFA; Merck) for 20 minutes at RT. To permeabilize the membranes, cells were washed 2 times with permeabilization solution (composition in Table 2.5). After discarding completely the supernatant, 1 µg of LTβ antibody (or the negative control) diluted in 100 µl of permeabilization solution was added and incubated for 1 hour on ice. Next, cells were washed two times using permeabilization solution, incubated with 0.5 µg of secondary

antibody in 50 µl of permeabilization solution, and incubated for 1 hour on ice in the dark. Finally, cells were washed 2 more times with permeabilization solution and resuspended with 500 µl of 10 mM NaN3 in PBSsolution.

2.4.3. Detection of membrane-bound LTβR ligands

Membrane-bound LTβR ligands were detected using the murine LTβR-hIgG (mLTβR-Fc) (Browning et al., 1997) and visualized using PE-conjugated AffiniPure F(ab’)2 fragment donkey anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories) secondary antibody, which was also used alone as a negative control. The mLTβR-Fc fusion protein and human IgG were provided by Jeff Browning (Boston University School of Medicine, Boston, USA). All antibodies were prepared in 2% normal mouse serum (NMS; Jackson Immunoresearch Laboratories) in PBS to prevent non-specific staining.

To detect LTβR ligands at the cell surface alone or in combination with T-cell markers, we applied an adapted immunostaining protocol based on the one described by Ansel and coworkers (Ansel et al., 2000). In detail, a suspension containing 1×106 _{cells was} transferred to each 5 ml round-bottom polystyrene tube (BD Falcon) and washed with 1 ml of ice-cold FACS solution (composition in Table 2.5). After washing, cells were centrifuged at 300 ×g for 5 minutes at 4ºC and the supernatant was discarded. To block Fc receptors prior to immunostaining, the cells were pre-incubated with Trustain fcXTM (anti-mouse CD16/32; BioLegend) at 1 µg in 100 µl of PBS for 10 minutes on ice. Cells were washed, the supernatant was discarded carefully, and 50 µl of 2% NMS containing 1 µg of mLTβR-Fc (human IgG or just 2% NMS in the negative control tubes) was added. After a 1-hour incubation on ice, the cells were washed two times with FACS solution. When indicated, cells were incubated for 1 hour on ice, in the dark with T-cell marker antibodies (Table 2.1) and then washed two more times with FACS solution. Next, cells were resuspended in 50 µl of 2.5 µg/ml donkey anti-human secondary antibody and incubated for 45 minutes on ice, in the dark. Finally, cells were washed 2 more times with FACS solution and resuspended in 1 ml of 10 mM NaN3 in PBSsolution. Where indicated, LTβ expressed at the cell surface was pre-blocked through 20 minute incubation with 1 µg of BBF6.BF12 in 200 µl of 2% NMS. Then, the cells were washed 2 times with FACS solution before the incubation with LTβR- Fc. To detect LTβ expressed at the cell surface, 1 µg of BBF6.BF12 monoclonal antibody in 50 µl of FACS solution (incubated at 4ºC for 1 hour) was used, and it was visualized using

FITC-conjugated goat anti-hamster (Armenian) IgG (0.5 µg in 50 µl of FACS solution and incubation for 1 hour at 4ºC, in the dark).

Negative controls and cells stained with each antibody individually were also prepared to adjust the FACS instrument settings and fluorochrome compensation.

Table 2.1. Antibodies used for flow cytometry analyses of T-cell markers and RANKL. Antigen Antibody

*

Clone Fluorochrome Concentration (mg/ml)

Dilution

Isotype Control Rat IgG2a, κ RTK2758 FITC 0.5 1/200

Isotype Control Rat IgG2a, κ RTK2758 PE 0.2 1/200

Isotype Control Rat IgG2a, κ RTK2758 PE/Cy5 0.2 1/400

Isotype Control Rat IgG2a, κ RTK2758 APC 0.2 1/100

Murine CD3ε Hamster IgG 145-2C11 FITC 0.5 1/200

Murine CD4 Rat IgG2b, κ GK1.5 FITC 0.5 1/200

Murine CD4 Rat IgG2b, κ GK1.5 PE 0.2 1/200

Murine CD8 Rat IgG2b, κ 53-6.7 PE/Cy5 0.2 1/600

Murine CD24 (HSA) Rat IgG2b, κ M1/69 APC 0.2 1/400

Murine CD25 Rat IgG1, λ PC61 FITC 0.5 1/400

Murine CD44 Rat IgG2b, κ IM7 APC 0.2 1/400

Murine CD69 Hamster IgG M1.2F3 FITC 0.5 1/200

Murine CD90.2 (Thy1.2)

Rat IgG2b, κ 30-H12 FITC 0.5 1/500

Murine CD254 (RANKL)