Download Macrophage Infection Protocol MEM (120707) Revised MG (111208)

C:\Documents and Settings\Loretta Garin\Local Settings\Temporary Internet Files\Content.Outlook\PE7VR7ZQ\Macrophage
Infection Protocol 110409 (3).doc
Macrophage Mycobacterial Infection Protocol
Original version from Michelle E. Maxson (W.R. Jacobs lab, 120707)
Revised by Michael Goldberg (Porcelli lab, 111208)
I. Growth Media for macrophage
A) Medium for growth of THP-1 cells (human myelomonocytic leukemia line)
RPMI-C (“RPMI-Complete”):
450 mL RPMI media (Invitrogen cat# 12633-020)
0.5% (or 2.5 mL) MEM essential amino acids (Invitrogen cat# 11130-051)
0.5% (or 2.5 mL) MEM nonessential amino acids (Invitrogen cat# 11140-050)
1% (or 5 mL) 1 mM HEPES (Invitrogen cat# 15630-080)
0.1% (or 500 µL) β-mercaptoethanol (Invitrogen cat# 21985-023)
20 µg/mL Amikacin (Sigma cat# A1174) (note: potency is 674-786 μg per mg,
prepare 1000X (20 mg/mL) stock based on potency).
10% (or 50 mL) heat inactivated Fetal Calf Serum (Invitrogen cat# 16140-071)
*Note, for plating THP-1 cells a day before infection, use RPMI-C with 50 nM of
phorbol myristate acetate (PMA; SIGMA), which "activates" the cells and
enhances their attachment.
B) Medium for growth of murine macrophage (primary and cell lines)
DMEM-C ("DMEM-Complete"):
450 mL DMEM media (Invitrogen cat# 11995-073)
0.5% (or 2.5 mL) MEM essential amino acids (Invitrogen cat# 11130-051)
0.5% (or 2.5 mL) MEM nonessential amino acids (Invitrogen cat# 11140-050)
1% (or 5 mL) 1 mM HEPES (Invitrogen cat# 15630-080)
0.1% (or 500 µL) β-mercaptoethanol (Invitrogen cat# 21985-023)
1% (or 5 mL) Pen/strep (Invitrogen cat# 15140-122)
10% (or 50 mL) heat inactivated Fetal Calf Serum (Invitrogen cat# 16140-071)
For primary murine macrophage (but not cell lines) also include 10% L929 CM:
L929 CM = “L929 conditioned media” (a source of murine M-CSF). To
prepare this, L929 cells (ATCC CCL-1) are grown in 100 mm TC plates in
10ml DMEM-C. Cells are split 1:5 every 3 days. When sufficient number
of plates are obtained, incubate for 7 days and then collect supernatant
(conditioned medium). This is filtered (0.2 μm), aliquoted and stored at
-70°C.
1
C:\Documents and Settings\Loretta Garin\Local Settings\Temporary Internet Files\Content.Outlook\PE7VR7ZQ\Macrophage
Infection Protocol 110409 (3).doc
II. Infection Media (no antibiotics)
A) Medium for infection of THP-1 cells
RPMI-I (“infection”) base media (prepare amount needed immediately prior to infection)
450 mL RPMI media (Invitrogen cat# 12633-020)
0.5% (or 2.5 mL) MEM essential amino acids (Invitrogen cat# 11130-051)
0.5% (or 2.5 mL) MEM nonessential amino acids (Invitrogen cat# 11140-050)
1% (or 5 mL) 1 mM HEPES (Invitrogen cat# 15630-080)
0.1% (or 500 µL) β-mercaptoethanol (Invitrogen cat# 21985-023)
10% non-heat-inactivated human serum (Gemini Bio-products cat#100-110)
*Note: for human serum, thaw 100mL bottle at room temperature, aliquot in
1-2 ml portions, and store at -20ºC.
Infection media should not be stored, and should be prepared freshly
immediately before doing infection.
B) Infection of murine macrophage (primary and cell lines)
DMEM-I (“infection”) base media (prepare amount needed immediately prior to infection)
450 mL RPMI media (Invitrogen cat# 12633-020)
0.5% (or 2.5 mL) MEM essential amino acids (Invitrogen cat# 11130-051)
0.5% (or 2.5 mL) MEM nonessential amino acids (Invitrogen cat# 11140-050)
1% (or 5 mL) 1mM HEPES (Invitrogen cat# 15630-080)
0.1% (or 500 µL) β-mercaptoethanol (Invitrogen cat# 21985-023)
10% non-heat-inactivated FCS (important to screen FCS lots to find those that
work well for this; our current lots I or U give good results)
III. Infection of macrophage with mycobacteria
1. Prior to day of infection subculture mycobacteria cultures at density needed to have an
OD 600 nm that will be between 0.6-1 at time of infection1.
2. The day before infection, lay down macrophage in 96 well plates (100 µl volumes) at 1 X
105 cells per well. Media to be used as follows:
THP-1 cells: RPMI-C + 50 nM PMA
Murine primary macrophage: DMEM-C 10% L929 CM (source of murine M-CSF)
Murine macrophage cell line (J774, RAW264.7): DMEM-C (without L929 CM)
3. On the day of infection, wash macrophage monolayers once with 1X RT PBS, and add
100 µl infection media (RPMI-I or DMEM-I). Place monolayers in incubator.
1
Inoculate 10 ml cultures with 2% (200 l) of M. smegmatis frozen stock, or 0.2% (20 l) of an actively growing, and turbid culture.
The cultures should then achieve the expected OD in approximately 1-2 days. Alternatively, the same method will take
approximately 7-10 days with slow growing mycobacteria (e.g., BCG).
2
C:\Documents and Settings\Loretta Garin\Local Settings\Temporary Internet Files\Content.Outlook\PE7VR7ZQ\Macrophage
Infection Protocol 110409 (3).doc
4. Prepare mycobacteria for infection as follows:
a. Pellet 3 ml of mycobacterial cultures at 2400G for 10 min.
b. Resuspend cultures in 3 ml fresh 7H9-C media2 (alternatively larger culture
volumes can be pelleted and concentrated into 3 ml).
c. Aseptically probe-sonicate bacteria using a 1/8’’ probe sonicator (Branson
sonifier 450), at power level 2 and pulse level 40, for 4 pulses (alternatively use a
cup sonicator attachment3).
d. Measure OD 600 nm of sonicated bacterial suspension using a 1:4 dilution into
1X PBS-0.05% Tyloxapol (if culture is very turbid, use a 1:10 dilution). When
measuring OD, one should consider readings from 0.05 to 1.0 within an
acceptable range.
e. Calculate MOI’s of 0.1, 1 and 5, using the following conversion: an OD of 1 = 3 x
108 cfu/ml. If necessary, dilute the bacterial suspension 1:10 or 1:100 to obtain a
density that allows desired MOI to be obtained with a volume that can be
accurately measured and delivered (in this case volumes from 20 to 100 µl work
reliably; routinely the volume of bacteria delivered should not exceed 10% of the
total culture volume).
5. Pipette bacteria into the infection media on macrophage monolayers.
6. Incubate infected monolayers at 37ºC in 0.5% CO2 incubator for 3 hrs.
7. Wash monolayers twice with warm (37 ºC) 1X PBS to remove extracellular bacteria.
8. Add RPMI-C/DMEM-C (containing 10 - 20 µg/ml gentamicin) to monolayer to inhibit
growth of extracellular/released bacteria in infected wells. Primary murine macrophage
must be maintained in DMEM-C 10% L929.
BCG-P [dsred] RIF
6230
BCG-P [dsred]
6020
SM [pG13]
9. At various timepoints, visualize wells by microscopy or lyse and harvest wells for cfu
plating.
MOI 0.1
MOI 1
MOI 5
SAMPLE OF TYPICAL EXPERIMENTAL SETUP: For our
experiment, the 96 well plates were infected as above.
2
7H9-C Bacterial growth media (1L): 4.7g Middlebrook 7H9 broth (500g, BD 271310), 10 ml 50% Glycerol (0.2 µm filtered), 900 ml
Milli-Q diH2O (18.2 mΩ) and 100 ml of oleic acid dextrose catalase (OADC) supplement (500 ml BD: 212351). Pass through 0.2 µm
filter.
3
The the cup sonicator should be set at power output 4 and duty cycle 60 for 3 pulses of 10 seconds each
3
C:\Documents and Settings\Loretta Garin\Local Settings\Temporary Internet Files\Content.Outlook\PE7VR7ZQ\Macrophage
Infection Protocol 110409 (3).doc
IV. Harvest of infected macrophage lysate for cfu plating
1. At various timepoints, aspirate media from infected macrophage wells, and add 100 µl of
sterile lysis buffer (0.05% SDS, w/v in H2O) to each well.
2. Incubate plates at room temperature approximately 5 min.
3. After pipetting up and down 2-3 times, transfer lysates to new 96 well plates for serial
dilutions. Duplicate wells are combined for a final volume of 200 µl.
4. Prepare 1:10 serial dilutions of lysates in the 96 well plate by pipetting 25 µl into 225 µl
of 7H9-C. For example:
5. Plate out 100 µl of chosen dilutions on 7H10 or 7H11 agar plates (plus any needed
antibiotics or supplements, depending on mycobacteria strain).
Alternatively; 10 µl can be spotted on square agar plates (36 spots per plate
decreases the amount of agar needed for each experiment)
6. Wrap agar plates in disinfected aluminum foil and incubate at 37ºC.
7. At 2-3 days (M. smegmatis) or 21 days (slow growing mycobacteria), remove plates from
incubator and count colonies.
8. CFU calculations are done using MS excel. Average duplicate or triplicate dilutions
plated.
# colonies / dilution = # colonies in 100 µl lysate (or per well, since 100 µl volume
of lysis and plating)
========================================================================
COMMENTS and FOOTNOTES:
Useful gadgets:
colony counting marker (Fisher, cat. #07-910-15).
Color cryobabies laser jet printer labels (USA Scientific, cat. #9187-1708) for labeling the
individual agar plates.
4