Biology Reference
In-Depth Information
10 6
cells/well in 4 mL avian osteoclast culture medium ( see Notes 4 and 5 ). It is
important to include a control well cultured with media alone (i.e., without
cells) to determine the background level of release from the matrix and also to
indicate the amount of LTBP1 in the matrix that is available for release by the
avian osteoclasts.
4mLPBS, and then seed avian osteoclast precursor cells onto matrix at 3
×
2.
C in a 5% CO 2 incubator then change media to
2 mL reduced serum avian osteoclast culture media. Culture for 48 h. For avian
osteoclasts, experiments are usually performed in the presence and absence of
1,25-dihydroxyvitamin D 3 , (10 -7 to 10 -8 M ) which stimulates the formation of
mature osteoclasts and stimulates their bone resorptive activity. At this stage any
test treatments, such as protease inhibitors, antibodies, growth factors, and so on,
can be added to the media to determine their effects upon release of LTBP1 from
the matrix by the cells.
Allow cells to adhere for 2 h at 37
°
3.
Harvest the 0-48 h culture media and transfer to 1.5-mL vials. Add protease
inhibitors (aprotinin 10
M and prefabloc
2 m M ) to the media samples. Microfuge at 16,000 g for 10 min at 4
µ
g/mL, leupeptin 2
µ
M , pepstatin 1
µ
°
C and transfer
C until ready for immunoprecipitation.
Add 2 mL fresh media (with or without test factors) to the cells and culture for a
further 48 h. This long culture period is used because optimal release of LTBP1
and TGF
supernatant to a clean tube. Store at -70
°
β
by avian osteoclast occurs on days 3-4 ( see Note 4 ).
4.
Harvest the 48-96 h culture media as described above.
5.
Prepare the ECM by washing the cultures twice with ice-cold PBS then adding
2 mL ice-cold RIPA buffer and incubating at 4
C for 15 min (protease inhibitors
should not be included at this stage because a plasmin digestion will subsequently
be performed to release any LTBP1 that is still bound to the matrix). Carefully
aspirate the RIPA buffer and wash a further 4 times in ice-cold PBS. Aspirate
the final PBS, and wash and air-dry the plates ( see Note 6 ). Plates can be stored at
-70
°
°
C for 1-2 d if required, prior to plasmin digestion and immunoprecipitation.
3.3. Immunoprecipitation of LTBP1 and TGF
β
In this procedure, labeled LTBP1 is immunoprecipitated from the media
samples to indicate how much was released into the culture medium by the
avian osteoclasts. To measure the LTBP1 which is still bound to the matrix, a
plasmin digestion is required since LTBP1 is crosslinked to the matrix and
requires proteolytic cleavage for release. Note: unless stated otherwise, samples
should be kept on ice for all steps described in the following procedure. A
refrigerated microfuge should be used for all centrifugation steps.
1.
Digest matrix preparations with 0.5 mL plasmin solution (0.2 U/mL) to release
LTBP1 and TGF
β
that is still bound to the ECM. Incubate for 1 h at 37
°
C with
gentle shaking.
2.
Transfer plasmin digest to a 1.5 mL vial and then add protease inhibitors
(aprotinin 10
g/mL, prefabloc 2 m M ). Microfuge at 16,000 g for 10 min and
transfer the supernatant to a clean tube.
µ
Search WWH ::




Custom Search