Issel Lim
7.021 Methods
Methods and Materials
Deriving Primary Macrophages
Macrophage Prep
A macrophage prep was performed in order to obtain the primary macrophages from two
strains of mice: C3H/HeJ black mice and C3H/HeSnJ black mice. The C3H/HeJ mice
possess a mutation in the TLR4 gene, and so were compared to the “normal” C3H/HeSnJ
mice.
Euthanization
The mice were euthanized using a carbon dioxide gas chamber.
Removal of the Bones
The bones were removed, cleaned with Dulbecco’s modified Eagle medium (DMEM),
and the marrow was flushed out. The cells were centrifuged at 1200rpm for 7 minutes in
a 50mL conical tube, and then plated in culture plates, incubated overnight in primary
macrophage media (350mL Dulbecco’s modified Eagle medium [DMEM], 10% Fetal
Calf Serum, 5mL Pen-strip, 5mL HEPES, 5mL L-glutamine through sterile filter)
.
Preparation of Bacteria
Bacterial Strains
Helicobacter bilis was the primary strain used in the infection studies of the
macrophages. It was grown on Petri dishes grown in vacuum jars in a 37oC incubator.
Ensuring Well-being of Bacteria
Two cultures of H. bilis was swiped two times and washed in a drop of saline on a slide
under a square coverslip. Using the 40x darkfield objective, the two Petri dishes’ strains
were compared based on motility and shape.
Preparing the Bacteria
After scraping the desired plate in four swipes, the bacteria were placed in 8mL of media
with no antibiotics (no Abs). 1mL of this mixture was transferred to a cuvette.
Referencing the spectrophotometer with the media alone, the optical density of the other
cuvette was then determined. An OD reading of 1 corresponds to 1.8 x 10^9 H. bilis
bacteria. One should provide for an infection of 100 bacteria for every one macrophage
cell.
Opsinization
To opsinize the bacteria, use a 1:2 ratio of equal volume of Phosphate Buffered Saline
(PBS) : BALB/C normal mouse serum of at least 100 microliters. Spin 0.5mL of bacteria
in an Eppendorf tube for 2-3 minutes at 10,000rpm in the centrifuge. Then aspirate the
media and put 100 microliters of PBS and 100 microliters of mouse serum into the tube.
Balance on a rotating wheel within the incubator for 15 minutes. Centrifuge the tube of
opsinized bacteria for 3 minutes at 10,000rpm. Aspirate the PBS and BALB/C mouse
serum. Resuspend the bacteria in 0.5mL of media and pipette a specific volume of this
into 10mL of media with no antibiotics. The volume should depend on the infection ratio
between cells and bacteria.
Infection and Incubation
Infection
Plate the macrophages in a 24-well plate on coverslips and incubate overnight. Then
aspirate the media from the cells meant to be infected and replace with 500 microliters of
media containing bacteria.
Centrifugation
Centrifuge the plate at 800rpms for 3 minutes in order to settle the bacteria onto the cells
and ensure contact for uptake in the infection study.
Incubation
Incubate for 1 hour.
Analysis of Infection
ELISA
Transfer the supernatants from each well to a corresponding well on a 96-well plate.
Either freeze these supernatants or analyze them using an Enzyme-Linked
Immunosorbant Assay (ELISA) to quantitatively test for various antibodies and growth
factors present in the supernatants.
Immunofluorescent (IF) Microscopy
Stain the living cells with lysotracker, incubating from 30 minutes to 1.5 hours. Then fix
the cells using 4% paraformaldehyde (PFA) with 300-500 microliters per well. Wash
three times in 5 minute intervals with PBS, then submerge in permeabilization solution
(90% serum-free media [DMEM], 10% goat serum, 1% of 5% saponin, 1% 10mM
HEPES). Prepare a parafilm with 20 microliters of selected antibodies per coverslip.
Primary antibodies used in this experiment were the LAMP antibody, anti-FC-receptor
antibody. Secondary antibodies were the anti-rat and anti-mouse antibodies. Each
coverslip was allowed to rest with the fixed cells in contact with the antibodies for 20-30
minutes. Then each coverslip was washed with PBS and positioned on a microscope
slide. They were stored in slide holders within the refrigerator or immediately viewed.