For analysis of int rac ellu lar comp on ents , cells were first fixe d in 3% p a r a fo r m a l d e h y d e for 10 minutes on ice, and then p e r m ea bi lis ed with 0.2% t rit on -X 100 in PBS for 10 minutes.
2.8.3 Flow cytometric analysis of intracellular levels of MHCII,
li and cathepsin proteinase expression
P e r m e a b i li s e d W M P T3 .3 or CaCo2 7.21 from each tre atme nt group were seeded into rou nd b o tt om ed 96-well plates at a density of at least 2 x 10^ ce l l s/ w e ll , wa sh ed repeatedly in PBS and then in cubated in 10% serum (rabbit or s h e ep -d ep en d in g on source of primary antibody) in PBS at 4^C for
10 min ute s. Cells were then in cubated at 4°C (agitating) for 40 mi nutes with
the f o ll o w in g antib odi es (detailed in table 2.2): TAL14.1, L243, LN2, anti- ca th ep s in B, M AB 44 2 , and C E l . l (Sealy et al, 1996). Antibod y supe rnata nts were added to each well at a dilution ratio of between 1:10 and 1:30. To de te r m in e levels of no n-specific antibody binding, 10% sheep serum was in cl u d ed as a control for the catheps in B group and the CII inc lu d ed as a contro l for all other groups. Controls in which each group was stained only with F I T C - c o n j u g a t e d second layer sera were also included.
F o l l o w i n g 40 m in ut es of incubation at 4°C with the primary antib odi es, cells were w a s h e d twic e in cold PBS and then left overnight, agitating and at 4°C, for the final wash. Second layer F IT C- co n ju g at ed antisera were then added
at a 1:500 dilution in 10% serum as follows: F IT C -c on ju ga te d donkey a nt i sheep Ig for the cat hepsin B group and FIT C- co nj u ga te d rabbit anti -m ous e Ig for all other groups. Cells were incubated for 40 minutes, agitating and at 4^C, and then washed twice in cold p h o s ph at e -b uf fe re d saline before p o s t fixa tio n in 3% PFA as described previously. Cells were finally re su sp en de d in p ho s p h at e -b u f fe r ed saline and kept on ice prior to, and during analysis. Analyses, of betwe en 2000 and 5000 events in each case, were p er fo rm ed using the Beckton Di ckinson FACScan flow cytom eter and co rre s p o nd in g FA CS ca n software.
2.8.4 flow cytometric analysis to determine rates of endocytosis
and exocytosis in WMPT3.3 using LY
In order to deter mine rates of endocytosis fo llowin g 120 hours of cytokine tre atme nt, 1 x 10^ WM PT3.3 from each cytokine tre atm en t group were cu lt ure d in 50pl Lucifer Yellow at 3mg/ml at 37^c for the fo llo wi ng time points: 180 mins, 60 mins, 30 mins, 15 mins, 10 mins and 5 mins (Levine &
Chain, 1992). Endo cytos is was then stopped by rapid cooling to 4°C. The
cells were washed twice with cold PBS and analysed by flow cytom etr y (as detailed previously). In order to determine rates of exocytosis, the lucifer yel lo w- tre at ed WMP T3.3 were then removed from ice and in cu ba ted at 37®C. Flow cytometric analysis was then perfo rm ed (as detailed pr eviously) at 3, 5, 10, 15, 20, 30 and 40 minutes, with the cells rema in ing at a cons tan t 37°C.
2.9 Immunoprécipitation of human and murine
class II components from B cells and epithelial
cells
2.9.1 Metabolic labeling
In order to deter mine total rates of MHCII synthesis in CaCo 2 or in W M P T3 .3 fo llowing 120 hours +/- cytokine tre atment, 4 x 10^ cells from each group were washed and in cubated in me th io n in e- fr ee D M EM at 37°C in
5% C O2 for 1 hour to remove free intra ce llu lar meth io ni ne. 18.5 MBq of
^^S-translabel were added to each group and the cells i ncu bat ed at 37°C for 3 hours. In corporation was stopped by rapidly cooling the cells to 4°C. Cells
3 5
were washed to remove excess S-label and then inc u ba te d in comp lete
med ium (RPMI with 5% FCS as detailed previously) at 37°C for a furth er 15 minutes in order to wash out any un -in c o rp or at ed meth ion ine.
2.9.2 Lysis
S-met hi oni ne labelled cells were washed twice in m ed iu m and r e
suspen ded in 1ml of cold lysis buffer (0.5% NP40, 150mM NaCl, 5mM EDTA, 150mM Tr is/ H C L pH 8.0) with 0.26 mg/ml Pefab loc and O.lmg/m l Le upeptin being included im mediately prior to adding to the cells. Lysis was conti nu ed at 4°C for 15 minutes. As with 0.5% TEA lysis of CaCo2, epithelial cells were lysed directly from the T ra ns we ll filters. Nuclei and Insoluble material were re moved by cen tr ifu ga ti o n and the s up ern ata nt
2.9.3 Pre-clearing and preparation of lysate supernatants
5 |il of nor mal ra bb it serum were added to the lysate su pernatants along with
50|li1 of a 5 0 % suspen sio n of P ro tein -A Sepharose, p r e-l oad ed with rabbit-
anti m o us e Ig. Sa mples were rotated for 1 hour with the Se ph ar ose at 4°C and then the S e ph ar os e- bo u nd no n -sp eci fi c material was r em ove d by br ief cen tr i fu g at i o n . The supernatant was transf err ed to a new tube and p r e cle ar ed with the P ro tei n- A Se ph ar ose a further two times.
The an ti b o di es used in the foll ow in g im m u no p ré ci p it at io n s were first co up le d to P r o tei n A-S eph aro se (preload ed with ra bbit-anti mo use Ig). 50|il of each ant ib ody supe rna tan t to be used, TIB93, L243 and TAL1 4.1 , was added to 20pil of Sephar ose su spension per lysate, and ro tated at 4®C for 40 min ut es. The Se p ha ro se beads were then washed twice with cold lysis bu ffer (as de ta il ed p r ev io usl y) and then finally r e- s usp end ed as a 50% s usp ens ion in lysis buffer.
2.9.4 Indirect immunoprécipitation
F o l l o w i n g p r e- cl ea r in g as detailed above, the cell lysates, W M P T3 .3 were i m m u n o p r e c i p i t a t e d sequentially with (1)TAL14.1, (2)L243 and (3)TIB93, using 20 \i\ of the ant ib od y /S ep h aro se conju gate per sample, for 1 hour. Sim il arl y, Ca C o 2 lysates were im m un o p re ci p it at ed with L243. The S ep h ar os e beads were washed twice with cold lysis bu ffer (as det ail ed pr ev io us ly ) and wash ed additionally with salt buffers as follows: one 1 m in ut e wash in normal salt buffer (lOmM tris /H CL pH8.0, 150mM NaCl and 0.5% NP 40 ), one 1 minute wash in low salt bu ffer (lOmM tr is /H C L pH8 .0 with 0.5% NP40) , one 1 minute in high salt buffer (lOmM t ri s/ H CL pH8. 0, 5 00 m M NaCl with 0.5% NP40), a further wash in normal salt b u ffe r and then a final wash in lysis buffer. All washes were car ried out at 4°C. Once the S ep h ar o se beads from each stage of the im m u n o p ré c i p i t a t i o n had been w a s h e d as deta iled previously, an equal volume of red u cin g sample
b u f fe r ( 2 0m M tr is /b as e, 1.0% glycerol, 2.3% SDS and 0.5% 2- M e r c a p t o e t h a n o l with br omo phe nol blue) was added to each, and the
m i x t u r e then b o i l ed for 7 minutes. 20|xl of each sample was run on a 12.5% p o l y a c r y l a m id e gel, dried, and ex po s ed to X-ray se nsitive film for 10 days.