EPISTEMOLÓGICA, DIDACTICA Y TEXTUAL
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2.1
Mice
The mice strains used in this study were F 5 R ag l7 ‘ (Mamalaki et al., 1993a),
N P47RagIV'(M am alaki et al., 1993b), NP47F5 (M amalaki et al., 1993b), G FPR agl / F5 (unpublished), p2m '/'R aglV '(Z ijlstra et al., 1990), as well as syngeneic R ag l'/H -2 ^ (Spanopoulou et al., 1994), gcNegRag-/-qq) (Di Santo et al., 1995), BIO Nude H-2’’’ and polyclonal control m ice BIO H-2^. All m ice were kept in conventional pathogen free, animal facilities at the National Institute of M edical Research.
2.2
Antibodies
ANTIBODY
SPECIFICITY
CONJUGATE
REFERENCE
53-6.7 CDS PE Pharmingen (San Diego,CA)
53-6.7 CDS APC Pharmingen (San Diego,CA)
53-6.7 CDS FITC Pharmingen (San Diego,CA)
GK1.5 CD4 FITC Pharmingen (San Diego,CA)
GK1.5 CD4 PE Pharmingen (San Diego,CA)
GK1.5 CD4 APC Pharmingen (San Diego,CA)
H57-597 TCR FITC Pharmingen (San Diego,CA)
H57-597 TCR PE CambridgeBioscience
(Cambridge, UK)
v p i i
TCR BIOTIN Pharmingen (San Diego,CA)M l/6 9 CD24
(HeatStableAntigen)
FITC Pharmingen (San Diego,CA)
1M7 CD44 B lO TlN Pharmingen (San Diego,CA)
1M7 CD44 PE Pharmingen (San Diego,CA)
16A CD45RB BIOTIN Pharmingen (San Diego,CA)
16A CD45RB FITC Pharmingen (San Diego,CA)
M E L -14 CD62-L BIOTIN Pharmingen (San Diego,CA)
H1.2F3 CD69 BIOTIN Pharmingen (San Diego,CA)
H1.2F3 CD69 FITC Pharmingen (San Diego, CA)
PC61 CD25
(lL-2 R a chain)
PE Pharmingen (San Diego, CA)
7D4 CD25
(lL-2 R a chain)
BIO Pharmingen (San Diego, CA)
F4/80 BIO (Austyn and Gordon, 1981)
Jo2 FAS
(CD95)
BIOTIN Pharmingen (San Diego, CA)
2.4.G2 FcR 11 NONE/BIOTIN (Unkeless, 1979)
N418 C D ll-C BIOTIN (M etlay et al., 1990)
M 5/114 MHC 11 BIOTIN (Bhattacharya et al., 1981)
R46A2 IFN-y BIOTIN (Abrams et al., 1992)
A N -18 IFN-y N O N E/BlO TlN (Prat et al., 1984)
XMG1.2 IFN-y PE Pharmingen (San Diego, CA)
XMG1.2 IFN-y FITC Pharmingen (San Diego, CA)
R3-34 R atlgG l
(Isotype control)
FITC Pharmingen (San Diego, CA)
R3-34 R atlgG l
(Isotype control)
PE Pharmingen (San Diego, CA)
2.3
Media
The culture m edium was Iscove’s M odified D ulbecco’s M edium (IMDM ) (Gibco BRL, Paisley, Scotland) supplem ented with 5% heat inactivated fetal calf serum (ECS) (Gibco BRL), 2x10 ^ M L-glutamine, lOOU/ml penicilin, lOOpg/ml streptomycin and 5x1 O^M P-mercaptoethanol (all Sigma, Poole, GB).
M edium for washing cells was air buffered IM D M (Gibco BRL) supplem ented with 0.21%NaCl, lOOU/ml penicillin, lOOpg/ml streptomycin (AB medium).
2.4
Determination of cell viability and cell number.
Trypan blue (Sigma) at a final concentration of 0.08% in phosphate buffered saline (PBS, 10.Ig NaCl, 0.32g KH2PO4, 1.4 4 9gN a2H P0 4 in H2O) was used to determ ine the
counter cham ber (BDH Ltd, UK) and light microscopy. D ead cells (stained blue) were excluded from counting.
To determine the absolute num ber of each thymic subpopulation, the percentage of each thymic subset, as determined by Flow cytomerty, was m ultiplied by the total number of thymic viable cells.
The absolute cells num ber of peripheral C D8 T cells in spleen or in Lymph Nodes
(inguinal, branchial, m esenteric and axillary, pooled together) was calculated as the percentage of positive cells in these organs, as detected by Flow cytometry, multiplied by the total number of viable cells. Finally the absolute cell number of peripheral CDS T cells was calculated as the total num ber of CDS T cells in the spleen plus two times the total number of CDS T cells in the Lymph Nodes.
2.5
FACS staining
2.5.1 Surface staining and analysis.
Antibodies used in flow cytometry are shown in Table 2.2. Prior to staining, 1x10^ cells were preincubated with unlabelled mAb to FcyRII/III (2.4G2) to minimise unspecific staining. C ells w ere w ashed and re-suspended in 5 0 p l ice-cold FACS buffer (PBS containing 0.1% sodium azide and 1% FC S), containing an optim al dilution o f the appropriate Abs. Cells w ere incubated for 30 m inutes at 4°C. Surface staining was performed with fluorescein-isothiocyanate-(FITC), phycoerythrine-(PE), allophycocyanine- (APC) or/and biotin (BIO)- conjugated m onoclonal antibodies (mAb). Cychrome 7 -PE streptavidin (Cy-7 PE, Caltag Laboratories, Burlingame, CA) or streptavidin -R E D 670 (Gibco BRL) were used as secondary reagents for biotin. For surface staining of peripheral blood lym phocytes, the same procedure was followed with an additional red-blood lysis step at the end. Erythrocytes were lysed after staining upon incubation at room temperature, with lOOpl 1% FACS lysing solution (Becton D ickinson). For each sample 2X10"^ live gated events were analysed. Cell surface antigens expression was determined by analytical four-colour flow cytom etry using a FACS C alibur Fow cytom eter (Becton Dickinson). Forward and side-scatter characteristics were used to exclude dead cells. FACS data were analysed with Cell Quest software (Becton Dickinson).
2.5.2 Intracellular staining.
Cells, either ex-vivo or after antigenic stim ulation or after PD BU / lonom ycin re stimulation, were harvested, washed and distributed to micro well plates. Surface staining was perform ed as described earlier (2.5.1). Following surface staining cells were washed and then fixed with lOOpl 1% paraphorm aldehyde for 20 min at 4°C. Cells were the perm eabilized upon incubation for 3 min at 4°C with lOOpl 0.1% NP40 (IGEPAL Sigma) in PBS. A fter washing in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) cells were stained with anti IFN-y mAh FITC or PE conjugated for 30 minutes at 4°C. As negative control cells were stained with IgG-1 PE or FITC conjugated Ab for 30 minutes at 4°C. A fter staining cells were washed and re-suspended in lOOpl FACS buffer prior to flow cytometric analysis. For each sample, 10"^ live- gated events were analysed.
2.6
FACS Cell Sorting.
Pooled spleen and lymph node cells from NP47F5 or F5 or BIO mice were stained with surface mAbs, as described in section (2.5.1). Cells were sorted on a MoFlo cell sorter (Cytom ation, Fort Collins). GFP positive cells were sorted directly according to green fluorescent intensity without surface staining, on a M oFlo cell sorter as well.