Fxtr~ction
~nrl
Concentration
from I'1lJf'inp
;.\ Senior
M~cro0ha~es
Iyrnphnodp'o"
Honore,
(Tn
of
Project
49CJ)
,l;,y
R ,i. 1 1
P;;lll
c;
~:;pr'in(~1
t
,i.
nVf:'r'~H)l
G,er
t pUn "1 v f'~ ,~ i l~ Y'
1c)c)1
Abstract
M ••••••••••••• M _ _ . _ •• _ · _ · •••• • ••••••••••
This
I' 81
project
e a s'j n g
nodes.
involved
c ul t u 1'1 n y
The
d
process
the
d her" e n t
was
based
rn do C r' 0 p hag e
cell s
f r" 0 rn
granulomas
using
collagenase
IH do C [' 0
P hag e
collected
P
I' 0
t
0
c
0
I a
for"
human
t pad
d'iseases
1a
that
has
known
antigen"in
a
the
of
spleens
cur's.
0
f ['
methods
m 0 use
s pl e ens
Some
model s
to
Lh e
0
protocol
m rn a r"l n e l y
used
treatment.
shown
such
C5/BL/6
and
These
in
rn
for
ph
extracting
an d l 'I vel'
data
on
f l el s 11 m a nl as i s
d eve i 0 pille n t
0
way
iny
n)
as
the
nodes,
du
Azar'
n by
not.
CU
rni c e
f r urn
has
of
[' S
was
tiH~
of
(
tile
a
no
exhibit
to
be
(1 - 5 ) •
and
regional
of
Res e a [' c i l l n
s u [' f ace
0
n "
cav I t y
p ['
"p [' e s e [l t.i
'Iyrnpilocytes
a
to
!Tl?j(![
p e ['I t oneal
by
d,
"i1 Y P e [' - e x p r" e s si
0
t e 'i n
11
9
to
act
'I e Ish rn a nl a si ~'.
sl iyht.
ulceration
the
shown
ulceration
6 - 9 ).
cel
as
cutaneous
been
E x p e [' I m ~~ n t al
leads
used
spleens
show
[' e s p 0 n s e
body.
but
arld
eventual
0 C
been
L ei?.f!rll?.rli .. ?
st'lmulate
mouse
ilas
strain
lals
to
foreign
lymph
Ka'la
and
al so
that
of
0
mlce
enlargernent
(10).
st.rain
mice
tl
t'l
t h e l rnrn un e
1 fl
a
rec
ex t r" a c ted
PECs)
ved
an
as
rnouse
swel
rec
phd g e s
as
01
t oj n
show
specific
in
0(:
P [Ya g e s
p u 1pol
sUCt1
Balb/c
nodes;
a 'I s 0
rn a c r"
'i s i n v
ayainst
mice
lymph
on
(cells
The
(si Leo f i n
o u ['I a b
o f
0
'le'Ishrnanidsis1n
v u 1 n e [' a bl e
infected
draining
00
0
on
ed
In
C I'
a
'I •
~! X t [' ern el y
f
I'
of
e x p e r'i rn e n t s i e a d 'j n y
lr1
'I ei silman 'I as 'i s.
The
t 11 e
e x p r' e s s 'I on
Expe['lrnental
model
III d
development
of
enlargement.
fout.pad
hyper-expression
of
10,
'f
a
on
their
(' e s p
0
mice
PECs
n se
and
that
(6-10).
r' e c
0
v e ('
['ece'ive
These
r f'
0
drug
mount
response
rni c e
immune
'I a c k
mice.
and
t he
Cyclosporin
A
similar
aJ s 0 1 a c k
an
tr"ea"t.ment
seve (. epa tho g e ni c
they
mount
effective
L '~',l?j,.9.,f.'i n f e c l 'i
l he
p('ophyl.itctic
immunosupresive
an
m
mice
Wiltl
(CsA).
to
the
r" e s p
0
n •
been
C5/BL/6
nse
0
r
n
0
shown
mice.
to
The
un t (' eat e d
the
0
Ba1b/c
th'B
have
h '1 P e (' - e x p r' e s ~;i
the
0
immune
I a
I"
on
t h ei ['
P f:.:Cs.
As
mentioned
above,
unprotected
of
peritoneal
increased
numbers
infection
progresses.
ineffective
immune
it Y P e f' - act i vat 'i
(11) .
d
nd
We
mac r"
0
0
know
n
Research
response
of
that
p hag e
la+
s
0
cytokines
act i vat i n g
In
have
lab,
we
mac (' 0 pi] age s pJ a y
[' ~! S
'i rl
been
t it e
0
r"
rec
mice
as
to
ti v e
mdY
as
Lhe
that
the
due
c (' eat e
an
Lo
pop ul a t i
gamma
0
r1 s
inter'fer'on.
an
mdcropages
determine
0
develop
be
c ei 'I
ILl,
( r"1A F)
between
trying
e f
suggested
p f'i a t eT
SUCr]
r act
relationship
has
0
mice
macrophages
these
m e i nap p f'
Interdependent
our
of
Balb/c
and
what
T
cel
s.
role
r i n e I' r e c t i v e i mm urI e
P 0 n s e t 0'1 e ish m a ni a si s •
Macrophages
rnof'phologies
these
cells
and
cells
an d
r' e d
p uJ p
rn a c r'
0
the
t it a t
it a v e
a[)out
0
been
III
the
01"
parts
di rfer'
the
PECs
the
f
of
the
s pi e e n
ex ten si vel '1
a c [.
0
P hag e s
speci)c
f ['
what
til e
pop ul
ct
stu di e d .
0
m
r"oles
I y rn p h
of
cavity,
t ion s
Due
node
these
ar"eas
b f'·it in,
peritoneal
a [' e
r rl e i ['
body.
accof'ding
mi c [' 0 gli ace 1 1 sol'
iver,
[' e 'J e a si n 9
known
can
The
rn de,' 0 phd 9 e s
di f f i cuI t '1) n
'iitt'leis
in
various
functions
populate.
Kuprfer
p it age s
populate
0
to
r
the
t i s sues ,
cells.
In
our
lab,
don eon
the majority of our macrophage
PEe san d
developing
lymph
J"' 0
a protocol
nodes,
immune
mac
we hope
response
to
This protocol
for'
to
J"'
act e d
I'
J"' 0
m the
determine
their
been
s pi e e r1 •
releasing.macrophages
better
has
from
role
BY
the
in
the
leishmaniasis.
is based on procedures used
adherent macrophages
granulomas by
p rl age s e)( t
research
From spleen
treatment
of
red pulp
to
release
tissue and
liver
tissues with cullagenase enzymes
(12,13)
M.c.!._t:. . ~.r._.j ...c.!.:.1 ...~. t1. ':?._t}).5L9....~.
1M ice -
A'11
breeding
.
_L.:..~...:L~.!!~ c.!.!).
B al b / c
x
C 5 I B L / 6 mice we J"' e
J . ~!:ll.."1.j. .9....r.~.
i n fee ti
wi t h 2 0 ;:
(".
subcutaneously
f e t 611
0
n a J"' y
in
the
stage promastigote has
in
0
n-
tile
P
J"'
0 t 0z0d
T i'l e
infected mice.
wi t h i " ], 0 - s t a ti
stage
C 611 F S e r' u fll
needl e.
we r' e
i s 01 ate d
r.r.1.0jgr· we [. e
L.
g r' 0 wI I I rl
Anima'l s wer'e
•
I' [. 0 fll
t il e
vi t r' ()
'infected
stage p r' 0 mas ti got e s i r1 j e c ted
left hind
been
Bal b/c fIlouse
footpad.
shown
str'airl
susperldedin PBS and del'iver"edin
f,;lauge
b r' e d i n our' own
facil ities.
spleens of
i n R P 1'1 I
and
to be
(14).
d.
r il e s t a t 'i 0 n a r"y
the most
infective
Lei s il rn a n 'i a
0.05 fill
\AI
e r- e
dose wi til a i./
'p
Cyclosporin
A
treatmenl-
C ~Ai n
oil.
Mice
wei g il t
t wi c e
a
day
p ['i
began
1
wer'e
day
0 [.
to
h
0
mice
g
u r"i n t e "
D.
f.. •
F.
G.
H.
I .
'2 +
.
P B SlQ£'..~ S ..G.J. 1 ::11 • / fIll'1 1'4aC1
8.06 mt1 r-.la"Hpo.
/ H 0
.'
'~·f·
2
1.5 rnM KH2f"J04
5 • 5 rni"l
- gl u c 0 ~:> e
2./ mr1 RCI
0.9 IIlr1 \",,211.1
.. 2
0.5 mM MgC1
6 H_O
2
L
Brought to a pH of /.3
6 '" 0 u g il L t 0 a n end v 0'1 u m e
'2 +
_<::..~-···""Ir.1 .Y.""·-"·-
and
v 211
~ •
orally
or
with
body
Treatment
~olution~ u~ed and
211 1
M. C?..~U...fj...~..~. P. y.J...~ ..'3....C::..(;;...9.....'....'?"
C.
treated
with
CsA
L ...rr.1.?j.9..L.i n f e c t i 0 n .
**un1e~~ noted otherwi~e
A.
f.:l.
wer'e
do~es or 150 m / kg
giverl
12
a t
Balb/c
.f..r.:..~._e.. fQ.E. !3._.?'_.~-
P [. epa [. e d
~
T il .j c>
0
d. S
r
0
1 u t "j
0
~torea at
40C.*~
nco n l ai ned :
1. 0 Oi'i t e [.
abo v e,
but
wit il 0 u t
st ep s
f"
G.
_t::.D..~.Y..!!I..~. ~L9.. l . ~. t i ..9...!~ ..~.-
minutes
before
wdf"fTled
to
(::;i grna
Co.
37
o
All
use,
C.
we [. e
by
sol u t i 0 n s
dissolving
Collagendse
we [. e
we r" e
use a
as
p r" e p d [. e d
enzyme
1,II,IV,
not e a i r 1
in
and
r' e
DPBSG
VII
'S
nom 0 [. e
dna
t han
30
previously
DNase
u"1 t. sse c t i 0 f)
•
I
10
A 9 a I.
§,F"~,Q_t~,.Q,,t:, -
,If».,!",r::"E",!?,,:t
fill
Cd
Co.
Then
a
2+
/r"ly
63.6
2+
s
01
u ti
f' r' e e
0
n
was
in
OP B S G
gr'ad ient.
p" epa r' e d
90
by
di s s
ml
solution 'was
01
v i n9
(SiglTla
pr'epar'ed
by
cornbi n'j ng:
I~
.
9
C.
rn a c r'
0
.a
p hag e
j
w e r' e
cell
n cub a t i
sal ine
eel I s
P (' e
was
to
u 'I t u r' e
0
rl
use,
b e c d. use
aurler'e
C
we r' e
to
was
per'
a
p 1 ace d j n
used.
to
f" e rn ai ni n 9
d.
s s t u d 'j e s
esterase
each
rn a c f"
every
dissolving
buffer
,'ernove
f t e ('
[1
ave
positive.
two
19
plus
gr'ovity
1.25
Illl
F'ilter'ed
S t ()
0
n
was
s
01
u t 'i
III 0
The
stock
f'ilter'
a.1M
s tor" e d
gently
PO
at
0
0
t han
spec
washed
at
paper-
Cur 1 l i
was
2
fill
h 0 u r'
w; t rl
All
adher"ent
0
9 5 I..
the sec e 'I
0
was
was
20.0
I'ernove
added
used.
f
P hag e s •
'j ~:.
characterist.lc.
The
to
a
1
bern a c ('
fic
solution
to
stated,
A I' t e r'
solut.ions
buffer
4
4
r' e
shown
cel'ls.
(solidJln
2
been
used.
ass u rn edt.
stock
O.OOlM CaCl
1 of
we r' e
rnacrophage
trlr"ougrl
III
w e 'I
cover
used.
other'wise
w e r" e
A
be
cui t u r" ~?
cell
microscope
Unless
s how n t h a t
a
well
have
each
0
to
S e r" urn
P hag e s
paraformaldehyde
3 • I
k
well
r
24
gldss
non-adrler'ent
was h
weeks.
p a (' t. i c"l e s •
C
0
glass.
add edt.
r' rn e d j n
square
,f.'"~,,r~a f ~,L,r,!l"~..J._,9.,,~..I.~,y',,,9_,~, ,E"j,_>.<_,~"t:"i"x,,~,Fresh
r0
slips
so'lution
Vl 0 U
af'e
1
sus pen S1 () n s
selectively
o f
III
(B e 'I co)
~>Ii P s
stock 90/. Per' c 01 s 01 u t '; 0 n
" / r1g f'r'ee DPBSG
1 x Cd.
h eat i n d. c t 'i vat e d F e t. 03,1 Cal
Previous
plates.
slip
m'l
?0 • 6
20.4 rnl
B
prepared
made
III
1
by
O.lM PO
sol u t 'i on
-'1-
was
so'lid
to
filtrate.
1 X.
A
use
by
(pH
par' a
r0
diluting
produced
1 ine
(kindly
Me di c a]
by
n
was
rn ad e
1:4 with
solution
0313/
an d
t fl e
C biB l / 6
p u ['1 I i c a t i
II 0 II S
al b u ITli n
(Sigma
I
d.
has
P e ci f i c
and
Roger
J.
N C) •
This
jus t
P [' "i
PO~
O.lM
0
t:.
r'
0
buffer
<{
t y pes
0
j'
just
f
bi
urn
II
dill 9
to
to
0
n
0 [
rn al
of
n
tu
the
cell
Ouke
becau~:>~:!
was
used
the
Balb/c
1a
d
f1 H C rn 01 e c uJ e •
1a
cap [' "i 'Ji c
was
dis s
r [\i
use.
s
d,
01
ci d
yed
was
1 : lOIn
done
to
I i rn rn uno glob ul 'ill.
diJ u e II t e o n t ai
lOX.
bind
t fl e
sol u t 'i
0
hybridorna
ce"]"]]i rle
u si n 9
priur
03131
the
Kurlander
shown
Ant 'i bod Y
diluent
( 8 SA)
0
of
con c e n t [' ate d
(1 b ) •
se
growth
been
h al
b
was
r" a 0 bi t
HI e
[' a b oi L
fl
lOrn 9 / rnJ
ed
s e I'
U fill
n
0 u vi IH:!
R F-' f1 1
1 64 LJ
s e r"
rTI
U HI
e di a
Co.)
An
anti - m u use
jug ate
marked
( p u ['
not
to
D'lJ uen t.
C
03137
by
portion
0 I'
n
serurn
p [' eye n t
doe s
0
by
0 u r' h am.
antibody
I mrn uno g"J 00 uJ "i n
I'aobit
ascites
donated
C en t e ['.
the
p ['i
41.:
stock
0
/.3)
were
COli
r" rn d."] de f1 y des 01 u t i
of
the
F a b ""
fl a sed
f ['
0
m
anti-La
t fl e
Fao
0
dY
Trlis
bi I I d
was
t
was
antibody
antibudy.
2
f'] u 0 I' e s e 'i n
P eJ - I' I" eeL)
antibody.
non s p e ci f"i c a 'J 'J y
use,
ant"i i:l
.{'-
0
fhe
III a
diluted
0
c y a n aLe
tot
9
c eJ J ~:>
use d
binds
Fab"~
c [' 0 P II age
1:10
1 sot hi
In
to
d.
the
Fragment
Fe
antibudy
c; e"' "I s •
Just
R a b bi t
S e [' u III
.t.Q..9_Lr.::..~. .~. !:.
_f.J....Y_()_f...~..~..~._t!u_l...t:. A. r:l...!:.. .l.J?..9....9. Y.. .?. ~_.~i..n. j...'.:~._y.
.M. ~ . (;-.!::..9..P.h. ~.y..~_ . ~. Lt f
.~.. ~...~.J. Q_l-
identify adherent
la+
standard procedure
.M.j. .<=:_.r.::..<?_.~..~..<?.pj...~.
under·
v·j ~i bl e l i 9 h t
D3 1 3 I
lymph node
for
.~ ..~..~lJli...r_I.. 9.:..U
T fl e
thi~
anti bod Y w a ~ u ~ e d t 0
matrophage~.
~taining
...9..r.I. - Co v e
r· ~li p ~
and U v l i 9 h t
determine
total
number of maerophages
u nde ,.
ight
luore~cent
UV
Fluore~cent
eel
s were
t
u~ed
·1
ab'~:,
(9) •
e x a rni ned
examined un de,·
at
4 OX
no,· ma l i i 9 h t
in examined area.
cells were counted.
con~idered
Our·
bot h
u ~i n 9 a U V rni c ,. 0 s cop ~~
Sli P ~ we,· e
~
wa~
we ,. e
(leis~).
fi ,.
g ..L .~. cJ...i:.~. ~.!.::..Iil..!I... :~.
Ia positive.
All
to
r [1 e n
"
2
c.
.F."..'.·..9...~..~ ..9...\,! .. ~
Ex
d C
t
for· L.~.J... ~..':.l...?jD..9
d e t ai ·1 s o l
.?:..9tl..~.T . §!..!:lJ~. lJ.l..<:l,..c::J. . <:>..P...tl...<:l,.g..~... ?..
e d c hex p e r·i rn e n t 03.1
K.~...?..~J.. t.~.
sec t i
0
collect
from
one
A.
r1ice
wer·e
B.
0 r· di n ·i n gpo pi L e d . l l y rn p h
C.
nor
two
sdcr·iriced
w e r· e
dish
containing
node
each
node s
wer·e
m·1 c r
0
In
1 I
bei'"ore
e x p e r·i rn e n t s
II,
IV,
gr·indingin
or·
treated.
1 Y rn P h
we r· e
perfusion
rnajor··ity
of
01
0
cells
ex per· i rn en t. s
In
10 fill
0
cat e d
erlzyrne
5 rnl
0
0
f
w e r· e
cell s
k nee
b e i li n d
a
r
pet r·
0
ViI
gauge
0 f' B 5 G
01
added
gr·oups
1·1 u ·i d
was
r·eleased
·1 Y rn P il
Centro i I"uged
to
e •
g·lds~;,
of
r el e
d
se
OPBSG
Lo
as
J P
r
8
ground
d
nd
9,
d u I·j n ~J
r· el eased
iflto
··1
10 •
floted
e x p e r·i rn e n L s
nee d
to
.J P i
and
was
Tissue
un its / fill
pOI·Lions
1- I
J PT
22
specii'"ic~:.
(see
til e n i n d ·i vi dual
i3 0 0
I'
and
1000 units/rnl
in
ups
.R. t:! ..~ ..'='..J.....t. . ?.. ).
10 nil
J P T 11 - 14,
OPBSG,
with
u n d l rl
wer·e
0
t r· a nsf e r· r· e d t
fr·osted
I n
tissue.
I n
resuspended
g r·
u rl d e r·
I. e
OPBSG at 4°C.
tilose
Collagenase
des c r·i bed
d
noLed.
(1
C'
s y (·i r 1 9 e
using
gr·
nod e···
asphyxiation.
nod e
w i til
s ·Ii de s i n to
node s
as
2
under
with
gr·ound
Collagenase
CO
per·fused
i n j e c ted
s cop e
cells.
10 rnl
was
Collagenase
Nodes
by
ex p e r·j [[I en t
we r· e
nodes
r· e rn 0 v e dan d
-I e g)
of
O.
lymph
I e r t
ll"lyrnph
E x per· i rn e n "t a ·1
pap e ( .•
or
rn e tho d
.X..f.:..9..!!! J. y.r.T).I-? . i.l.
until
suspension.
n o\d e s
pellet
we r· e
at
4
o
9 r·
°u n d
C,
and
Collagendse
~. 01 u t i on.
E.
C (~ll s
we r· e i n cub
do
ted
dL
:3! 0 C i n
a
5;:'
(; 0 2'
e r1 vi r·
0
rlITl e n L •
p
p
f.
roll
0
w "i n 9
the
r" el e a s e
washed using
cell s
nil
Ca/Mg
f r" e e
reel "I s
free
The
i r1 DPBSG
C a / r'l 9
0
D PBS (1.
pellet was
(1.
e a/ Mg
enzyme
activity.
c e "11
1f
s
we r" e
then each
1
rnl
t
be
0
test
C a / i'1 9
fuged
cells
resting
using
transfer pipette.
wit.h Ca/Mg
+
H.
The gradient with
centr
1 0 ;:
f e t 03.1
c e"1 "I s we r" e
1 f
pellet was
free
r10 t
t
e r u rn
0
be
resuspended
ad "i en i: ,
t.he
layer was
cover
The cover
were
resuspended
(f C S)
layer" and
removed
for"
t w"i c e
in RPMl
RPMl
1640
c 0 u n t1 n g •
s e p d. r" ate d b Y 9 r" a di e nt,
in
in
gradient.
Celis wei" e washed
DPBSG and
~:;
y r"
[1 ai L
L0
resuspended
surface of percol
C al f
01
f1 9
of per"col
at 1800 9 at 4°C.
on
o r"
suspension was
carefully layered on 8-10 rnl
so"1ution.
per" c
s was
T 11 e
•
enzyme
use d i n was i1
s e p .;\ r" ate d on
0 PBS (1
in 10
t i1e p r" e s e (1 c e of
on
group of cel
f r" e e
n '> w e r" e
r"esuspended
Collagenase
DP B~:; G was
f r" e e
0
T i1 e nth e sec eli s we r" e
dependent
ion s .
sus per 1s "i
DPBSG by centrifuging down
c e n t. r" i lug e d dow nay a in.
("eact I on
t. i1 e
1640 +
101
t i1 ~~ n
feS
for
cQun t.i ng.
1.
Cells were counted on hernocytorneter,
desired concentration.
W
81 "1 e d p"l ate s
for"
then
a d her" e nee
diluted
to
sarnples were added
"i n cub a ti a r1 .
t.a
+"
Specific
t.he
guidelifles
of
eacil
exper·iment
ar·e
r·esults •
e.
. ~~.. P...~ . r.:. .t.r.f.1.~Jl...t....._. ~. L. _._.J,.
r·li c e :
'2 g r· 0 ups 0
L .!Tl<:\j()r·i n f e c t i
r
0
'2
n
u n p r· 0 t e c Led
w e r· e use d .
B al b / c
mi c e
i n
we e k
4
0
f
Pr·ocedur·~~ :
..................................................
r h e ·1 y fTl P h nod e s w e r· e gr· 0 u n d wi tho u t e n 2: y met r· eat fTl e n tor·
incubation, and placed orl Per·col gr·adierlt.
Cel·ls wer·e
Inc u b d. ted, was h ed,
[ i xed wi t h 1 I.. par· a [0 r· rll al d e h y de, a fl d 1 f
stained.
f\ .~.. ?IJI.t..?.... :.
No macrophage
ex arni nat ·i on .
cells
were
observed
under
microscopic
.t.:.. ~..P.~..r.::..::i.. f!1.~.r.:I. . t. ....... ~.e.T. 2
r·, ice:
..............................
'2
rTla.,j()r:i rl
groups of '2 unprotected
r e c t i 0 n w e r· e used.
Pr·ocetiur·<·~
................................. ,..
Balb/c
rnice
in
week
5 of L.
:
In
thIs procedure Collagenase VIi at 1/0 units/ml +
DNase I 20 units/rnl
in 8 rnl DP8SG.
~'iuspension Wd.S
inculJat~"d
for· 10 Hli nut. e s be I' 0 r· e C a / r"'i g I' r· e e was r1 •
() t il e r· wi s e i den t i cal
to Ex. JFT 1.
R{:~su·lt.s:
f', gelatinous mass developed
washing.
No cell s obser·ved wi til
I
art e r
I i r· s t
micr·oscop·ic
Ca/r1g Iree DPBSC,
e x a IT! ina t i 0 r 1 •
..E.,..>.<..p._~_LJ.f!1.l:!.. Q...~_ .......~..E.T.. . . .::i...!..4.
Mice:
Each experiment
L .!Tl.:=tjgr·i n fe c t i 0 rl •
P r·
0
c e d u r· e
~:>
used
4
groups
of
2 mice
in
week
:
...............................................................
In these e x p e r·j rn e r 1 t. s, 1 / 0 u nj t s ;' rnl
substituted for Collagenase VII.
C o·j ·1 age n a s e l l
4-6 of
'p
T
!
i';;.esults:
Gel f 0 f' Ifl e d
er'r'vneously was
a sin
round
J PT 2. T he C a / r1 y l f' e e D PBS G s 0 '1 u t i v n
to be stock lOx ~:,oluLion.
fhe SOIUl.lOrl
fl ad bee I I I abel e d 1 x
and the h y P e f' ton i c
I Y si s an d the f 0 f' mat 'i 0 fl 0 I' the 9 el .
s vi u t i 0 n
c au s e ci
c ei '1
,~"~"P'..,~_,!::,j.I!1_~,n ..~,_,_".~"E.I_.__?,,,:'.,
i'1i ce:
2 groups of
fTI<:l,jor:i fl I' e c t i 0 fl •
2
unprotected
Balb/c
mice
at
week
5
of
L.
p.r..'.. ()<:,:,,~,g ..':l,.r:Y~.
In l his e x p e f'i rn e n t 0 fl e 9 f' 0 u po' f i y rn p f 1 nodes was t f' eat e d
wi til Coil agenase I I 1/0 U/fIll and tfle Ol:fle[
tf'eated wi t.il
Col '1 age r 1 a s e 1 V ( no t. e: n ei l i 1 e [ , sol u l i 0 [ 1 con t ai rl e d f.) r·J a s e 1) a L
sam e dil u t i 0 n •
E a c h t est y f' 0 u p W d. S 9 f 0 U 11 d, t f' e d. ted wi til
enzyme solution, and incubated for' at 3/'\; 10f' 10 fll'irlutes.
x
5
fhen groups were washed, cul tured at ~.O 10
cells/rnl RPMi +
1 0 "/ F C S '1 n 2 <1 well c el 1 wei 1 pl ate; til e n ~, t d.i fl e (j •
Hesu'lts:
.......... ...................... .
-
Coil age n a s e l l l [' eat. fTl e n t p [" 0 d u c e ci an ave ['age 01'
adhe['ent c e l l s / f i e l d witil 68.9';:' of cells beillg Id.+.
C 01'1 age r 1 a s e IV t [' eat m e [1 t P [" 0 due e (j an a v e r' aye of
a d il e f' e n t e e II s / 'Ii e 'I d wi t h 6 5 • :j:r.: 0 I' cell s b e i n 9 I a~' •
3
4. 2
Mice:
2
fTl<:l,j()f:i n
groups of 2 unprotect.ed
r e c t ion w e f' e use d •
Balb/c
fIllce
at
6 of
week
L.
P.,I:,,(),..~,~.gyr:~~,,~,
In l
to t.hose
t.o t.f'eat.
ti'l e p [' 0 c e
ili sex p e [' i me [1 t, '1 Y III P h nodes we [' e t [' eat e d i den t i c 0.'1
used In Ex. JPT 5; except, collagenase IV was used
bot.h yf'OUpS of mice.
'This was done t.o detefrni[le 'if
sse 0 ul d de I i v e [' con si s ten t r' e sui t;, •
Results:
...............................................
Whefl ce'lls examined undef' fluQ['escent fTlicf'Oscope,
ins u Y f i ci e n t cell s f 0 [' ace u r" ate data coli e c t i 0 [1 W e r' e round.
;'1.,'11 slides con t ai n e d l e s s t il a n 1 a d 11 e f' e n t cell /
ri e 'I d on
a v e r" age •
Hice:
2
[!l<:l,j()r:
groups of
in fe c t i 0 rl.
2
unprotected
Balb/c
mice
at
week
8
of
L.
p
f' . [:.gr;~...tJ. ',J..r:. ~ . . ~.
J n t hi~;, e x p e [. 'i men t '1 Y rn p il 11 0 0 e s w e r' e I' e m 0 V ed, t [' eat e d
witil Collagenase iV as in Ex. JPT 5,
incubated FOf' 10
Ini nut e s, g I" 0 U n d again using cl e an f [ 0 s ted si i des. Tile
remainder of the procedure was ioenlical to that of
[:',x. JPT 5.
ror
Ayaininsuff'icient numbel's
collection or 121 expression
of cells
data.
couid
be
obse['ved
,~.,?<"P_,~ ..r.::.,::i"'!'"E!!,,,~l_,1..,,,,,,,;J.,,f'_,T,,_,,,,~,,.:'.,
M.i
ce .:..,
2 unprotected
a n 0 2 C 5 I B L / 6 mi c e
ITl'?,jgr.i n I' e c t i 0 n •
Balbjc mice, 2 CsA protected
wa I' a usa d •
All fll 'i c e wa I' e i n
Balbjc
wa e k i:3
rnice,
0 I' L.
Pf"oceouf'e:
....................................................
Lymph nodes were removed, and grouped by type:
U 11 P I' 0 t e c ted. C sA. 0 [' C 5 I' •
i n t hi s a x p e f" i rna 11 i:.l Y rn P h n 0 0 e ~,
WEll"e pe['fused with 5 rnl Collagenase 11 at 800 U/ml.
fi'lell
9 [' 0 uno sus pen si 0 n s weI" e i n cub ate 0 I' 0 I' 1 0 mi nut e s and L il e n
wasiled wi til Ca/Mg l'I'ee DPB5G.
Cell s wel'el ayel'ed 011 Pel'col.
a II dee n t. f' i I' u g e d •
T [1 e con c e n t I' ;;~ t i 0 n 0 f c e 'II s .j n e a c h f'< P ", I
~, 0 '1 uti on was d 'il u ted to 1. 8 xl 0 () c e ') '1 s / rnl.
1 ml sam pi e s we f' e
tl"ansfal'f'ed to pI'epaf'ad walls in 24 we'll p·late.
Plates wel'~"
centrifuged at 150 gravities to sediment. celi s.
Plates were
'i II cub ate d I' 0 f' 2 h 0 U I' S • I'i xed t 0 s l i des u si n 9 1 I:'
P d I' a f 0 I' III a'l d e h y des 01 u t i 0 n , and t [Ie nco vel' e ci wit h P 8 51 0 I"
later examinat.ion.
The cells were not. If stained.
f:~: . ~!...?...~. ".!. ... ~.... ~~....:..
Unprotected Balb/c mice were counted at an average of
:3 • 2 a 0 h e I' e II t cell s / I i e ) d , i n 5 0 I i el d s •
C~A protected Balb/c mice were counted at an average 01'
8 . 0 a 0 h e 1- e n t cell s / I i al 0 i n 5 0 I'i el os •
C 5 7 B L / 6 rni c ewe I' e c 0 u n ted a t a vel' age 0 r 9. 1 a d h e I' ell t.
cell s; f i el o i n 50 I i el d
s.
.M,.i ..,<.: ..,~.,.,:..,
2
i n Ie c 1. 'i
e.
G r 0 LJ P s or 2 C sAp I'
0 II weI' e
use 0 .
0
t e c 1. e d
rni c e i n
week
6
of
L •... I:I.l..<:l,j,().r:,
f,'.. .9.,..<.::.,~, ..si,.~J.I.'.,~,. :.
P I' 0 C e d LJ I' e i ci e n t.i cal
t. 0 E:. x. ,J P 'j 8 was LJ sed un t i I
o r cell s •
5 u s pen ;;i on s o l
two g I' () ups we I' e pool e dan 0
diiuted to 2.01(10° celi ~>/ml RPr-il solution.
6 sl ides
p I·epaf'eo.
di) uti
then
we I'e
011
H.esults:
.'\ v e ,. a 9 8
I i el d c 0 u n t s
cells/tield
C
0
to
_t::_>,<..p.._~_F.5.r.!1_~.n_~ __ ._.~_e
el 1 c 0 u n t o t 4. 3 a d il e ,. e n t c 81 1 s / i i el d, but b 0
f di r fe,' e n t s 1 'i des a v e , age d f ,. om • 8 ad he," e n t
b.3
adherent
cells/fi~ld.
. T. ._._:1,..Q
,"1i c e :
';' yr'oups or 2
irli'ection we,'e and
L .rll<:l,jqri n 1 e c t ion
P r"
e d u r'
0 C
........................
~~
unprotected Balb/c mice in week 9 or
1 yroup of CsA prutected mice at week
we,' e
/
of
use d •
:
Lymph nodes were removed, yround in 10 fill 01 DPBSG +
Collagenase IV 800 U/rnl.
::3uspension c , we,'eincubated ror' 10
rn i r1 u L e ~;"
r- e 9 " 0 u n d and inc u bat e d f 0" 1 0 m 0 ,'e rni nut e s •
C e '11 s
we,' e t i1 e n was h e d wi L il C a / "1 9 i' I' e e [) P B :::> G •
Al
sus p e 11 si 0 n s wei' e
cornbined, and washed with Ca/Mg Free [)PBSG.
Pellet was
" e sus pen d e d i n 2 rn '1 C a / "1 9 f " e e D PBS G •
1 fill 0 r sus pen s'i
I aye ,- e d on per' c 01 and c e n t ,·i rug e d •
A I" t e r- was i1i n 9 cells
twice. pellet was resuspended in 4 fill RPMl + lOl fCS.
::> u s pen ~,i 0 r 1 was d '11 ute d t. 0 4 x 1 0 6 c ell ~, / fill
1 1111 s a rn pi e~,
0
n w ;;.I, ~:>
we,' e
L r" d n s r e " " e d to p" epa r" e d cell w e '11 pI d t e •
1 he pI d. t. e w d, s
C e '11 s w 8 r- e ri x 8 d to
c e rl t r-i rug e d t il e n i r1" u bat e d 1 0" '2 il 0 u r' s •
s l i des by t r' 8 d t m e 11 t wi t. h 1 t:: par' a f 0 "rn al d e h y de, t. hen s t. d, i i1 e d
using fluorescent antibody staining procedure.
Result.s:
12.9 ad h e I' e n t
Cell s we r- e 0 b s e " v e d at 11.4
Ave
,"age
cell
number' in
in 30 rield inspections.
counted was 12.0 adherent ceil s/fielci.
.............................................
cells/field
4 slides
_t::..>.<.P..~..!.=j.r.!1. ~_r.].,_t_. __ .~.F.'..I_._._.:1,...!.
fvli c e :.
.•....
unprotecteci Balb/c
inl'ectiorl wer'e used.
mice
in
week
18
or
L. "rllo.,jgr:
P........................................
r- 0 c e ci u " e :
11 P oli t e 211'1 y rn p h nod e san d t i1 " e e '1 y rn p h n 0 ci e s f r' 0 III t i1 e
lowe,' b a c k s 0 f t i1 r' e e 0 f t i1 e ani rn a'l s w e r' e " e rn 0 v e d f I' 0 rn rn ice •
L y rn p i1 nod e s we,' e all 9 ,. 0 u n d tog e t i1 e r ' i n 1 0 rnl D P 6 S G •
Suspens'ion was cer1tr'ifugeci to pellet.
The pe'l'let wa~>
resuspended in 1.5 rnl DPBSG + 18/0.5 units of collagenase
(Tilis t,'eatrnellt was appr'oxirnately 1000 U Collagenase/rnl of
pel'leted ce·l·ls).
Ce'I'ls wer'e incubated ror- 30 rn'inutes at
:3 / 0 C •
A t 5 fIli nut e i n t e " v 211 s, the cui t u [' e s we [' e ,. e rn 0 y e d r r' 0 rn
i "1 cub a to,'. YO,' t e xed I' 0" '2 sec 0 11 d san d , ' e pi ace d i n inc u bat 0 ,. •
.'\fte"irlcubation. 8 rn',
t 0 c u'l t u r' e d n d was il e d •
Cd./ M 9
I " ee
D PBS G .
T 11 e '1
of cililleci Ca/r1g I,'ee DFBSG was added
C e'l 'j s we,' e w ,~ s i1 e d do 9 doi n wi til 1 0 rnl
C
ells
we,' e
,e sus pen d e d i n
1 '2
fill
R P r1 1
+ 10l FC:::).
Suspension was tr'ansfer'r'ed to pr'evious'ly pr'epaf'ed
c ~:1 'il cuI t. u [' e p'l a t. e i nO. 95 flli al i 4 u a t. s •
C e 'i
s we [' 12
centri fuyed for' 5 minut.es t.o set.t.le cells on glass, then
inc u bat 12 d for' 2 h 0 u r" s •
A I' t. 12 [' inc u baLi 0 n c 121 'I s w 12 r' 12 was h 12 d
arlei fixed using 1;;: par'dlor'mdldehyde solut.ior).
Re~ults:
Cells in RPMl solut.ion were count.ed
One cover sl ip WdS completely counted at
o t fj 12 r' s l i p s we [' 12 no t com pi 12 t 12 'i Y c 0 u n t. 12 d ,
simildr' counts.
a L 2.0 x 1 0 6 cell s /ml •
625 c e 'II s •
T fH~
but appeared t.o have
,~">,<"p""~,_cj"I!l"t:!.,r.:!"t.,_,_",~,e,.:r.:"",,,,:J,,,,?,,,=.,'
1'1 ice:
01
2 CsA prot.ected and 2
L'J!j::l,jgr:infection wer'e
unprot.ect.ed
used.
Balb/c
mice
in
week
B
Pr ...............................
ocedur'e:
,
p 0 pI i t 12 a l l y mph nod e S w 12 r' 12 p 0 0 led, 9 [' 0 u n d, c 12 n t. r'i f u y e d
d s i n Ex. JPT 11.
Ce'l'l suspensi orl WdS tr'edted wi t.h
C 01 'I a 9 ~1 n a S 12
1 s 01 u Li 0 n a t 1 0 0 0 U / ml p 121 'I e i.. v 01 u me.
Aft e r' 3 0
minut.e incubdt.'iofl with vor'texing dt. 5 minute int.er'vdls,
liquid was t.r'ansrer'red to f"r'esh cenLr'ifuge t.ube and washed 2
times wit.h Ca/My Free DPBSG.
2.5 ml of Ca/My Iree DPBSG was
add 12 d t. 0 t.i s sue mas s r' 12 m ai n i n 9 f r" 0 rn 12 Ij Z Y met. r' eat. III e r j t. •
1 h 12
:':>u:':>pension was vOI"texed lor' 5 minutes. WdSfled Ollce and
combined wit.h other cel IS.
Cell:':> were resuspended in 2 ml
C a /1'1 g r r' e 12 D PBS G •
Cell con c e n t r" a t. ion was de t e r' min e d. d, n d a 1
nli ~,arnpie waslayer'ed on per'col and separ'ated.
The ot.her' 1
m 1 :':> a m pi e was c 12 n t. r'i I' u y e d to p e I I 12 l.. a Ij d r ' 12 sup 12 n dedi n 1 2 rn "I
RPMl + 10l FCS.
After percol t.reat.ment and washes, t.hat.
sample was also resuspended In 12 ml RPMl + 10l PCS.
A U her' e n c e C ul t. u r' e s w e [' e p [' epa I' e d: 1. ~2 rn 1 0 f peL' c 0 'I
t. I' eat e d cell s we r' e di yi d ed, 6 well :':> at l u l l :3. 4 x lOt) cell s / ml
x
and 6 well:,:> dt 1.2 l0o cell s/ml.
12 1111 s~mp~e 01' untr'eated
c 12 '11:':> we r' e i r j cub ate d. 6 weI 'I ::, at Full )'. 0 1 U
cell:':> / m 'i and 6
wells at 3.5 cell s/ml
£j
Resul ts:
..................... _...................
Cover'
slips
we [' e
counted
and
r' e s ul t. s
'listed
below.
6
Per' c 01
t r' 12 ate d cell s a t :3.4 x 10
con C 12 n t. r' a t.i 0 n a v 12 r' aye (i
19 cellS/field examined.
Percol
t.reaLed,cell :':>rat. 1/2
x
()
COl Ice n t. r" a t.i 0 n a v 12 r" age d Bee 'II s /
Fi 12 'I d .
)' • 0 l O u n s 12 p a [' a t. 12 d
cells aver'aged 18 cells/Field.
Tfle unt.redt.ed cells at 1/;~
concent.I"ation were counted at 11 cel'ls/window.
Due to the
siflli 'I ar'i t.y or r'esu'l t.s 0 r cell s sepdr'd,ted on Per'col and t.ilo~>G
not t r' 12 ate d,
i L was dec ide d not t 0 use Per' c 01 :' 12 P d r' d t i 0 n
In
f"ut.ure experiment.s.
>
.e. I.........! ...~.
.~..~..P... ~...r..j.~..~Jl...h..... ~.
/'1i ce :
..............................
:3
u~ed.
E!al b
9 [.
0
up ~
of
2 C51Bl/6.
mi c e l n week 4 of l. ..f.Tl.a.,jgl·j n fee t i 0 n w e [. e
2 C~A treated Balb/c. and 2 untreated
Ie.
Pr'ocedur'e:
.........................................
Lymph node~ were removed and pouled by type.
Combined
wei 9 h t s 0 1'1 Y rn P h node 9 [. 0 ups we r' e de tel" rnl r1 e d •
E a C 11 L e ~ t
gr'oup of 'lyrnph node~ wa~ yr'ounciill lU rn'l DPBSG and
centrifuged to pellet.
Each pellet was resuspended in 1 rnl
of 1000 unit/rnl Collagena~e 1 fur every rny of lyrnph node
weight.
Cell s weI' e cui t u r' e d i 11 e n Z y f1l e sol u t i 0 n I' 0 I" 3 0
rni nut e san ci v 0 r" t e xed at 5 rni nut e i n t e [. v a'l ~ d u r"i n 9 tIle
inCUbation.
After' illcubation the ceil susperlsions wer·*..~
washed twice with Ca/Mg free DPBSG.
Pellet wa~ re~u~pended
i n 2 rnl RPM 1 + 1 0 l
F C San d v 0 [. t e xed i 0 I' 5 rn i nut e s •
2 fill rn 0 roo ~.~
01' RPMl + 10l FCS was added to each ~afllple.
Ce'll~
wel'e
,
. . x
6
I rnl
counted and each ~arnple wa~ dlluted to 2.0 10
cell s
o . 9 5 rnl a l i qua t ~ 0 f s a rn pi e s we r' e t [. all s fer' I . e d t u p (. epa r' e d
wei 1 ~I n cell cui t u [. e plate, c e n t ('i f u y e ci then 1 n cui) ate ci for' '2.
1/'2. Ilour·s.
NOI,-adller'ent cell s wel'e wdsheci Ir'ofll sl ips.
I~;. e rn 0.1 n '1 11 9 cell s we I'e r i xed
to s l i p s u si I I 9 1 t.: P a [. 0. for' rn a'l d e h y ci e
l. hen 1 F s t ai 11 e d •
Results:
Counts of total cell
Un pI" 0 tee ted
CsA pr'otected
C5lBl/6
Gr'oup
unp [. 0 t e c
'"
I nurnber' o r
. . . . L... i!l.C: ..I,!.i:l ate d
I
ted I
4
E)a.,.. I .. i?Lc:........
J ...
I
I
i??..lI?Lc. .........L...
C~A
L [. eat e ci
4
i n . ~16rnph
9.6 10
c·
6
t:i. 0 xl"u,
node
samples
3.g X 10(
adher·ellt cell s I
.... L. i n :2? .... I.Lel.. (;j...?..L..
I
5
I
................L
I
1/
I
..J...... . . . . . .
we I '1 s
t:.§L.t.3.I"I§...L . .
63
I"
1.;:\+
I
cells/I
c eli s .. 1 fi.t:?10 ........ L
2U.Ol
0.2
35.2l
.68
............................. I/[:3.~Q/ . ...1 .......4... ~?:2.J
Due to dn dccident. ollly one slip/sarnp'le could be
d a to. s h 0 u'l d not b e c on si ci e r' e d ext [. e f1l el y [. eli a bl e •
coullt.ed
l'1i
ce: .
..
9 I'
used.
Ualb/c.
'2.
ups of Illi c e i n week 5
C5/Bl!6, '2. unprotected
0
I
I
L • [11. i3j(l { or
1 11 fee t i 0 n w e [. e
Balb/c, 2 CsA protected
and
Er·(?~.e.glJr·
P r'
e:
c e d u r' e i den t
suspensions w e r" e all
+
lOZ
0
FCS
instead
of
1 c a '1
t. 0 Ex.
dil ute d to
x
6
2.0 I0
JPT l~ was used except,
,x . 1' O' 6 eells/mi
.
,.
2 .0
In
t.he
RPi11
.
Results:
. ............ _...................... .
Count.s of t.otal cells 'in 2 lyrnpll r19Ge
x
Urlpr'otected Balb/c
5.0 lO
CsA protected Balb/c = 2.7xlOI
x
CS/SL/6
2.2 I0'
sarnple:
Gr'oup
n u rn b e r" 0 r wells I d d II e r' e r1 t c el '1 s
. . . . . . . . . . . .. . . . ..J .... i.r~.c;.lJt:l?.Le. ci .... . . . ...............1 ......:1. rl.......'?:. ?I:.l e 1 cj s .....1
infectedl
I
27
!:l.:.~)i:>.Lc;. ..J . . ........................1.: .................................................L .
CsA
I
I
t.r·eatecJ I
1
I
65
t301l?1~.J
(;?ZE.lL.19J .................................. J ..................................... 1.. . . . . . . § /
I cf~lls/ I
cell s ....1. ..ri ..e... i.. d ........ L
I
I
6 6 • 7 X.I..l.~ .. :l,.. ...........L
Z
1-3.+
96.91:
2. 6
J
... 1........X~?tl ....... 1
................... 1. ..............................................
P.:i.. ~.g..':J....~...~j._<?.fl.
.E'.F.Q..9._J. .~. f!1...~...... .:i..r.~. . . . g.~._'f~J.. Q..P.J. !.}..g..._....~.l.}. ~....... P.... r.::.g_ . ~..9_.c;:..g..J.. .:.. '
1.
Miss
2.
Later
L .r!l;;,tJ.o..r:·
labeled
solutions.
studies
suggest
p r' 0 due eve r' y
I n c 0 n t. r' olin 0 del
ex p e ['i mer) t s.
0
mi g [) t
r
0
t
[) a v e
0
Balp/c
mac r' 0 p [) age s •
few
p r'
that
c
The
0'1 •
been
due
low
to
mice
infected
These
mice
nurnbe[s
of
t h e l ow
with
used
were
ce'llsin
r) u m be r' s
0
c el "I ~:>
r
available.
3.
due
a'll
t
0
Insufficient
01 0
w
r0
w e ci
treatment
con c e n t r' d t i
[ ' i n cub a t i
0
0
nor
n.
of
tissue
e r) z y m e
m .j g h t
[) a v e
0
with
collagenase,
r " i n s u f f .j cj e n t
c a use d
the
inc
0
ti
fII
e
n s 'i s t e r1 t.
[' e s u'l t s .
4.
have
E?
V0
x pl ai n
12-14.
Note
0
rn a c r
the
p hag e s
t.r·ed,t.rnent.
for
0
f [.
wit.h
0
n s i r)'1 ate rOo
of
enzyme
e x p e [ i men t s,
treatment.
rn i g h t
This
coul d
rOo e s u'l t s •
suggest.s
allows
that
sus per) si
homogeneity
bet t e ['
ci [.) e rOo e n t
rn d c r
t e xi n g
improved
Data
d.
['
p
[1
that
the
age s
rn
protocol
extraction
0
f
consistent
0
the
the
t [) e l y rn p hoi d
collager)dse.
dnd
rn u rOo i n e
release
used
p
or
in
Experiments
selective
0 ~
1 y rn p !)
'li tea'l
relatively
t i s sue
Al though
see III s
on'l y
t
study
0
JPT
or
node s •
large
numbers
bed e pen den t.
coli dgenase
1
0
wa~:>
n
U
5 e di n
might
the
r'i n a 1
produce
de v el oped
5imilar
con c e r 1 t. [. a t i
0
al "
e a r"l i e r"
0
w e di n
Not
n s,
enough
rn a cr'
0
p tl age s
the
preliminary
'1 eve', s
0
f
Ia
sh
0
ul d
rn d
C
r'
0
a 1 '1
0
w
phd g e s
macrophage
use d i n
t'issue
arld
data
f () [.
a
rn 0
in
releasing
0
s.
n g e [.
la
0
the [.
coIl a
Q
end 5 e
u 5edi rl
hi ghe r'
t. ["' eat men t
t. han
the y
lymph
node
expression
t.
[.
e
.j
rl
P hag e
types
w e [. e
de p t h
stu d y
portion
of
n dj n
and
the
vi t r'
0
f
n s:
h
0
we v e
the
other
studies
f'
in
bet wee rl
T hi s
mice.
protocol
0
0
differences
n u m b e r"
susceptible
leishmaniasis
d
con cl u s 'i
significant
fTl do C [. 0
and
in
d r" a wan y
0
nan d
c Y tom e t e [. y
eel
0
f
suggests
resistant
role
f '1 ow
on
coIl e c ted
e x p r' e s s 'i
leishmanidsis
'1
toe 01,
e x p e ['i rn e n t s •
data
was
0
re5ul t5
wi t. h
and
p [.
" y mph
p [.
0
t. 0 c 01
nod e
diseases.
The
could
be
of
'1 y
al so
III P h 0
'i d
Thanks
A -;per i ril
th,l.nk
[')rojpri':
~,l.I('re",C,
and
in
YOlJl-
put
d
he;,<,t
you
hoth
to
0,
0
NAnry
helped
me
teric:hing
RehforOlJ7o
in
And
so
mAny
you,
othpr
srienc:p ..
Jay
w;;ys ..
YOll
Thank-;