3. Fixable viability dye eFluor 506 (eBioscience, San Diego, CA).
4. Fc block (anti-mouse CD16/CD32 monoclonal antibody
(clone 93)) (eBioscience, San Diego, CA).
5. Sorting buffer: 5 mM EDTA + 5% v/v FBS in PBS, pH 7.4.
Filter sterilize using a 0.22 μm filter.
6. A flow cytometer with sorting capacity, equipped with at least
four laser lines (405, 488, 561, and 633 nm) (see Note 6). We
utilize a BD FACS Aria cell sorter. Samples are sorted through a
100 μm nozzle.
7. 8-chambered cover glass (Ibidi, Germany).
8. Poly-L-lysine.
9. Phosphate-buffered saline (PBS), pH 7.4: 1.058 mM
KH 2 PO 4 , 5.6 mM anhydrous Na 2 HPO 4 , 154 mM NaCl.
10. Alexa Fluor 647 conjugated phalloidin (ThermoFisher Scientific, Waltham, MA).
11. 4
0 ,6-Diamidino-2-Phenylindole, dilactate (DAPI, dilactate)
(ThermoFisher Scientific, Waltham, MA).
12. A confocal microscope equipped with at least four laser lines
(405, 488, 594, and 633 nm), and high magnification oil
objectives (40Â, 63Â). We utilize a Leica SP8 spectral confocal
system (see Note 5), with image acquisition via Leica Acquisition Suite X software, and subsequent image analysis carried
out with Volocity software (Quorum Technologies, Ontario,
Canada).
3 Methods
3.1 Construction
of Single Fluorescent
Reporter
M. tuberculosis Strains
1. Using primers that contain appropriate unique restriction sites,
PCR amplify from M. tuberculosis genomic DNA the selected
promoter region of the transcriptional reporter to be made (see
Notes 7 and 8).
2. Gel purify the PCR product for further downstream cloning.
3. TOPO clone the purified PCR product (see Note 1), and verify
the sequence.
4. Using the sequence-verified TOPO clone, digest and ligate the
promoter into the GFP-containing vector pGFP-N, and transform into E. coli TOP10.
5. Conduct diagnostic digests on isolated plasmids to verify successful generation of the reporter construct.
6. Transform the newly generated reporter plasmid into
M. tuberculosis to obtain the fluorescent reporter
M. tuberculosis strain, and characterize as described in Subheading 3.3 below.
370
David Giacalone et al.
4. Fc block (anti-mouse CD16/CD32 monoclonal antibody
(clone 93)) (eBioscience, San Diego, CA).
5. Sorting buffer: 5 mM EDTA + 5% v/v FBS in PBS, pH 7.4.
Filter sterilize using a 0.22 μm filter.
6. A flow cytometer with sorting capacity, equipped with at least
four laser lines (405, 488, 561, and 633 nm) (see Note 6). We
utilize a BD FACS Aria cell sorter. Samples are sorted through a
100 μm nozzle.
7. 8-chambered cover glass (Ibidi, Germany).
8. Poly-L-lysine.
9. Phosphate-buffered saline (PBS), pH 7.4: 1.058 mM
KH 2 PO 4 , 5.6 mM anhydrous Na 2 HPO 4 , 154 mM NaCl.
10. Alexa Fluor 647 conjugated phalloidin (ThermoFisher Scientific, Waltham, MA).
11. 4
0 ,6-Diamidino-2-Phenylindole, dilactate (DAPI, dilactate)
(ThermoFisher Scientific, Waltham, MA).
12. A confocal microscope equipped with at least four laser lines
(405, 488, 594, and 633 nm), and high magnification oil
objectives (40Â, 63Â). We utilize a Leica SP8 spectral confocal
system (see Note 5), with image acquisition via Leica Acquisition Suite X software, and subsequent image analysis carried
out with Volocity software (Quorum Technologies, Ontario,
Canada).
3 Methods
3.1 Construction
of Single Fluorescent
Reporter
M. tuberculosis Strains
1. Using primers that contain appropriate unique restriction sites,
PCR amplify from M. tuberculosis genomic DNA the selected
promoter region of the transcriptional reporter to be made (see
Notes 7 and 8).
2. Gel purify the PCR product for further downstream cloning.
3. TOPO clone the purified PCR product (see Note 1), and verify
the sequence.
4. Using the sequence-verified TOPO clone, digest and ligate the
promoter into the GFP-containing vector pGFP-N, and transform into E. coli TOP10.
5. Conduct diagnostic digests on isolated plasmids to verify successful generation of the reporter construct.
6. Transform the newly generated reporter plasmid into
M. tuberculosis to obtain the fluorescent reporter
M. tuberculosis strain, and characterize as described in Subheading 3.3 below.
370
David Giacalone et al.