15. It is important that if multiple 24-well plates are being infected,
each plate is allowed a similar amount of time for bacterial
infection. Take note of the time each plate was infected and
initiate at least the first wash step for each plate following 1 h of
infection.
16. To reduce position effects associated with evaporation, we have
found it useful to stack the plates and place an empty plate on
the top and bottom of the stack. Additionally, for a 6-day
infection, we place the plates in an open plastic Ziploc
® bag
with a moist paper towel to act as a local humidifier. For
consistent data, avoid using the two outside rows and columns
in a 384-well plate and one outside row and column in a
96-well plate as these wells are the most prone to evaporation
and edge effects.
17. To maintain the sterililty of the coverslips, we have found it
useful to use a vacuum to transfer single coverslips to each well
of a 24-well plate. In a biological safety cabinet, attach a tube to
a vacuum source and a sterile 1 mL pipette tip to the other end
of the tube. Apply the vacuum and the pipette tip will hold a
single coverslip that can be transferred to a well in a 24-well
plate.
Acknowledgments
Research in the Abramovitch lab is supported by a grant from the
National Institutes of Health (R01AI116605) and AgBioResearch.
Research in the Olive lab is supported by grants from the National
Institutes of Health (R21AI148961 and R21AI146504), the
Department of Defense (PR192504) and AgBioResearch.
References
1. Rohde K, Yates RM, Purdy GE, Russell DG
(2007) Mycobacterium tuberculosis and the environment within the phagosome. Immunol Rev
219:37–54
2. Ralph P, Nakoinz I (1975) Phagocytosis and
cytolysis by a macrophage tumour and its cloned
cell line. Nature 257(5525):393–394
3. Tsuchiya S, Yamabe M, Yamaguchi Y,
Kobayashi Y, Konno T, Tada K (1980) Establishment and characterization of a human acute
monocytic leukemia cell line (THP-1). Int J
Cancer 26(2):171–176
4. Fejer G, Wegner MD, Gyory I, Cohen I,
Engelhard P, Voronov E, Manke T, Ruzsics Z,
Dolken L, Prazeres da Costa O, Branzk N,
Huber M, Prasse A, Schneider R, Apte RN,
Galanos C, Freudenberg MA (2013)
Nontransformed, GM-CSF-dependent macrophage lines are a unique model to study tissue
macrophage functions. Proc Natl Acad Sci U S A
110(24):E2191–E2198. https://doi.org/10.
1073/pnas.1302877110
5. Jordao L, Bleck CK, Mayorga L, Griffiths G,
Anes E (2008) On the killing of mycobacteria
by
macrophages.
Cell
Microbiol
10
(2):529–548.
https://doi.org/10.1111/j.
1462-5822.2007.01067.x
6. Wang GG, Calvo KR, Pasillas MP, Sykes DB,
Hacker H, Kamps MP (2006) Quantitative production of macrophages or neutrophils ex vivo
using conditional Hoxb8. Nat Methods 3
(4):287–293.
https://doi.org/10.1038/
nmeth865
182
Benjamin K. Johnson et al.
each plate is allowed a similar amount of time for bacterial
infection. Take note of the time each plate was infected and
initiate at least the first wash step for each plate following 1 h of
infection.
16. To reduce position effects associated with evaporation, we have
found it useful to stack the plates and place an empty plate on
the top and bottom of the stack. Additionally, for a 6-day
infection, we place the plates in an open plastic Ziploc
® bag
with a moist paper towel to act as a local humidifier. For
consistent data, avoid using the two outside rows and columns
in a 384-well plate and one outside row and column in a
96-well plate as these wells are the most prone to evaporation
and edge effects.
17. To maintain the sterililty of the coverslips, we have found it
useful to use a vacuum to transfer single coverslips to each well
of a 24-well plate. In a biological safety cabinet, attach a tube to
a vacuum source and a sterile 1 mL pipette tip to the other end
of the tube. Apply the vacuum and the pipette tip will hold a
single coverslip that can be transferred to a well in a 24-well
plate.
Acknowledgments
Research in the Abramovitch lab is supported by a grant from the
National Institutes of Health (R01AI116605) and AgBioResearch.
Research in the Olive lab is supported by grants from the National
Institutes of Health (R21AI148961 and R21AI146504), the
Department of Defense (PR192504) and AgBioResearch.
References
1. Rohde K, Yates RM, Purdy GE, Russell DG
(2007) Mycobacterium tuberculosis and the environment within the phagosome. Immunol Rev
219:37–54
2. Ralph P, Nakoinz I (1975) Phagocytosis and
cytolysis by a macrophage tumour and its cloned
cell line. Nature 257(5525):393–394
3. Tsuchiya S, Yamabe M, Yamaguchi Y,
Kobayashi Y, Konno T, Tada K (1980) Establishment and characterization of a human acute
monocytic leukemia cell line (THP-1). Int J
Cancer 26(2):171–176
4. Fejer G, Wegner MD, Gyory I, Cohen I,
Engelhard P, Voronov E, Manke T, Ruzsics Z,
Dolken L, Prazeres da Costa O, Branzk N,
Huber M, Prasse A, Schneider R, Apte RN,
Galanos C, Freudenberg MA (2013)
Nontransformed, GM-CSF-dependent macrophage lines are a unique model to study tissue
macrophage functions. Proc Natl Acad Sci U S A
110(24):E2191–E2198. https://doi.org/10.
1073/pnas.1302877110
5. Jordao L, Bleck CK, Mayorga L, Griffiths G,
Anes E (2008) On the killing of mycobacteria
by
macrophages.
Cell
Microbiol
10
(2):529–548.
https://doi.org/10.1111/j.
1462-5822.2007.01067.x
6. Wang GG, Calvo KR, Pasillas MP, Sykes DB,
Hacker H, Kamps MP (2006) Quantitative production of macrophages or neutrophils ex vivo
using conditional Hoxb8. Nat Methods 3
(4):287–293.
https://doi.org/10.1038/
nmeth865
182
Benjamin K. Johnson et al.