3 Methods
3.1 Growth
and Preparation
of Mycobacteria
for Cellular
Infection Assay
1. Inoculate frozen M. tuberculosis strains in the required volume
of fresh pre-warmed (37
C) 7H9-OADC-Tw medium (less
than 50 mL) containing the appropriate antibiotic (see Note 1),
to reach a starting OD 600 of 0.2, in a sterile and ventilated
125-mL Erlenmeyer.
2. Incubate at 37
C, 5% CO 2 without shaking for 7 days.
3. Measure the OD 600 and dilute the 7-days old M. tuberculosis
culture into 50 mL of pre-warmed 7H9-OADC-Tw plus
required antibiotic (see Note 1), to reach a starting OD 600
of 0.2.
4. Incubate at 37
C, 5% CO 2 without shaking for an additional
7 days (see Note 16).
5. Measure the OD 600 and dilute the M. tuberculosis culture into
100 mL of pre-warmed 7H9-OADC-Tw plus required antibiotic (see Note 1), to reach a starting OD 600 of 0.2, in a sterile
and ventilated 250-mL Erlenmeyer.
6. Incubate for 3 days at 37
C with shaking at 200 rpm.
7. Pellet bacteria by centrifugation at 3750 Â g for 5 min at room
temperature.
8. Resuspend in 35 mL of pre-warmed PBS and pellet bacteria by
centrifugation at 3750 Â g for 5 min at room temperature.
9. Repeat step 8.
10. Resuspend bacteria in 5–10 mL of freshly prepared and
pre-warmed RPMI-FBS.
11. Centrifuge at 220 Â g for 2 min to pellet clumped bacteria and
use homogeneous supernatant for infection (see Note 17).
12. Calculate the bacterial concentration by measuring the optical
density (O.D.) and the relative fluorescence units (RFU) of the
bacterial stock suspension using a spectrophotometer and the
microplate fluorescence reader, respectively. Determine the
bacterial concentration referring to the pre-established relationship, such as 1-unit O.D. is corresponding to 10
8 CFU/
mL bacteria concentration, and the CFU ¼ f(RFU) linear
relationship previously established for the used fluorescent
bacterial stock (as described below in step 14). Dilute bacteria
in RPMI-FBS to reach the needed MOI (see Note 18).
13. Optional: mycobacterial batch titration.
(a) Prepare series of tenfold dilutions of bacteria in PBS using
a 384-well plate (dilutions from 1 to 10
À10 fold).
(b) Record fluorescence levels (RFU) of each dilution using a
microplate fluorescence reader.
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Nathalie Deboosere et al.
3.1 Growth
and Preparation
of Mycobacteria
for Cellular
Infection Assay
1. Inoculate frozen M. tuberculosis strains in the required volume
of fresh pre-warmed (37
C) 7H9-OADC-Tw medium (less
than 50 mL) containing the appropriate antibiotic (see Note 1),
to reach a starting OD 600 of 0.2, in a sterile and ventilated
125-mL Erlenmeyer.
2. Incubate at 37
C, 5% CO 2 without shaking for 7 days.
3. Measure the OD 600 and dilute the 7-days old M. tuberculosis
culture into 50 mL of pre-warmed 7H9-OADC-Tw plus
required antibiotic (see Note 1), to reach a starting OD 600
of 0.2.
4. Incubate at 37
C, 5% CO 2 without shaking for an additional
7 days (see Note 16).
5. Measure the OD 600 and dilute the M. tuberculosis culture into
100 mL of pre-warmed 7H9-OADC-Tw plus required antibiotic (see Note 1), to reach a starting OD 600 of 0.2, in a sterile
and ventilated 250-mL Erlenmeyer.
6. Incubate for 3 days at 37
C with shaking at 200 rpm.
7. Pellet bacteria by centrifugation at 3750 Â g for 5 min at room
temperature.
8. Resuspend in 35 mL of pre-warmed PBS and pellet bacteria by
centrifugation at 3750 Â g for 5 min at room temperature.
9. Repeat step 8.
10. Resuspend bacteria in 5–10 mL of freshly prepared and
pre-warmed RPMI-FBS.
11. Centrifuge at 220 Â g for 2 min to pellet clumped bacteria and
use homogeneous supernatant for infection (see Note 17).
12. Calculate the bacterial concentration by measuring the optical
density (O.D.) and the relative fluorescence units (RFU) of the
bacterial stock suspension using a spectrophotometer and the
microplate fluorescence reader, respectively. Determine the
bacterial concentration referring to the pre-established relationship, such as 1-unit O.D. is corresponding to 10
8 CFU/
mL bacteria concentration, and the CFU ¼ f(RFU) linear
relationship previously established for the used fluorescent
bacterial stock (as described below in step 14). Dilute bacteria
in RPMI-FBS to reach the needed MOI (see Note 18).
13. Optional: mycobacterial batch titration.
(a) Prepare series of tenfold dilutions of bacteria in PBS using
a 384-well plate (dilutions from 1 to 10
À10 fold).
(b) Record fluorescence levels (RFU) of each dilution using a
microplate fluorescence reader.
664
Nathalie Deboosere et al.