Materials and Reagents

Note: All work with live Mtb must be performed in a Biosafety Level 3 (BSL3) facility according to institutional standards of practice.

1. Macrophages, either primary macrophages, such as C57BL/6 bone marrow-derived macrophages (BMDMs) or a macrophage cell line (such as RAW264.7) 
   Note: BMDMs can be isolated as described (Banaiee et al. 2006; Nagabhushanam et el., 2013). RAW264.7 cells can be purchased from ATCC (ATCC, catalog number: TIB-71 ).
   
2. L929 cells (ATCC, catalog number: CCL-1 )
   
3. Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Gibco^®, catalog number: 11965 )
   
4. Fetal Bovine Serum (FBS) (heat inactivated) (Life Technologies, Gibco^®, catalog number: 10082147 )
   
5. 1 M HEPES solution (Life Technologies, Gibco^®, catalog number: 15630-056 )
   
6. 200 mM L-glutamine (Life Technologies, Gibco^®, catalog number: 25030-081 )
   
7. Penicillin-Streptomycin solution (10,000 U/ml) (Life Technologies, Gibco^®, catalog number: 15140-122 )
   
8. Phosphate buffered saline (PBS) (Life Technologies, Gibco^®, catalog number: 10010-023 )
   
9. Eight well Permanox chamber slide (Thermo Fisher Scientific, Nunc Lab-Tek Chamber Slides, catalog number: 177445 )
   
10. Eight well chamber coverglass (Thermo Fisher Scientific, Nunc Lab-Tek Chamber coverglass, catalog number: 155411 )
    
11. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
    
12. Bovine serum albumin (BSA) (fraction V) (Thermo Fisher Scientific, catalog number: BP1600 )
    
13. Detergents: Saponin (Sigma-Aldrich, catalog number: 47036 ), Triton- X100 (Sigma-Aldrich, catalog number: X100) and/or Tween-20 (Thermo Fisher Scientific, catalog number: BP337 )
    
14. Primary antibodies to detect host cellular markers 
    For example, recycling endosomes and early phagosomes can be labeled with mouse anti-transferrin receptor (anti-TfR) antibody (Life Technologies, Invitrogen^TM, catalog number: 136800 ); Late endosomes and lysosomes stain with rabbit anti-LAMP1 antibody (Abcam, catalog number: 24170 ). 
    Notes: 
    1. If Mtb infected slides are to be removed from the BSL3 for imaging, the antibodies chosen need to work after fixation cum sterilization methods like long fixation as mentioned below in step A11, note a. Some of the commercially available antibodies may loose recognition or weakly recognize their epitopes after long fixation. 
       
    2. It is critical that polyclonal antibodies were not raised in animals given Freund’s adjuvant, as then they will directly recognize Mtb in addition to whatever cellular marker they were raised against. All antibodies should be tested to verify that they do not directly recognize Mtb. 
15. Secondary antibodies for immunofluorescence
    Secondary antibodies are available against different species and in different colors and user may choose depending on the primary antibodies being used. They are adsorbed against multiple species to minimize species cross reactivity during immunostaining. For example, Goat anti- mouse Alexa 594 (Life Technologies, Molecular Probes^®, catalog number: A11032) and Goat anti-rabbit Alexa 594 (Life Technologies, Molecular Probes^®, catalog number: A11037 ).
     Of note, Mtb exhibits autofluorescence, with an emission maximum at 475 nm when excited at 405 nm, and thus are visualized by many DAPI filters (Patiño et al., 2008). Therefore, secondary antibodies should be chosen that do not fluoresce in this range. 
    
16. Lysotracker Red DND-99 (1 mM stock in DMSO) (Life Technologies, Molecular Probes^®, catalog number: L-7528 )
    Note: Lysotracker dyes are available in different colors and one may choose depending on the color requirement. 
    
17. Dextran (TexasRed, 10,000 MW, Lysine fixable) (Life Technologies, Molecular Probes^®, catalog number: D-1863 ) (make 25 mg/ml stock in PBS, stored in dark in -20 °C)
    Note: Dextran is available in different colors and molecular weights and again one may choose depending on requirement and desired goals of the experiment.
    
18. Vectashield mounting media (Vector Laboratories, catalog number: H-1000 )
    
19. Middle brook 7H9 broth (Difco, catalog number: 271310 )
    
20. Albumin-dextrose-catalase (ADC) (BD, catalog number: 212352 )
    
21. Oleic-albumin-dextrose-catalase (OADC) (BD, catalog number: 212351 )
    
22. Nail polish (clear)
    
23. Immersion oil (Microscope 50CC Immersion oil) (e.g. Nikon Corporation, catalog number: IB-MA-MXA20234 )
    
24. 4% paraformaldehyde solution in PBS (see Recipes)
    
25. DMEM complete media (see Recipes)
    
26. DMEM/L929 complete media (see Recipes)
    
27. L-Cell conditioned media (see Recipes)
    
28. 2% BSA in PBS containing 0.1% saponin (see Recipes)
    
29. 2% BSA in PBS containing 0.1% triton X-100 (see Recipes)
    
30. 7H9 complete media (see Recipes)
    
31. Fixative (see Recipes)
    
32. Blocking solution (see Recipes)

Equipment

1. Spectrophotometer (measure the OD₆₀₀ i.e. optical density at wavelength of 600 nm of the mycobacterial cultures using cuvettes)
   
2. Disposable 1.5 ml cuvettes (Perfector Scientific, catalog number: 9003 )
   
3. Disposable sterile filter system (500 ml, 0.22 µm pore size) (Corning, catalog number: 430758 )
   
4. 30 ml square media bottles (Thermo Fisher Scientific, Nalgene, catalog number: NE/2019-0030 )
   
5. 50 ml, 15 ml falcon tubes (with plug seal caps) (Corning, catalog numbers: 430052 and 430290 )
   
6. Coverslip (22 x 50 mm, thickness#1, 0.13-0.17 mm) (Thermo Fisher Scientific, catalog number: 12-545C )
   
7. Centrifuge with swinging bucket rotor for spinning down bacterial cultures (for example, Beckman Coulter, model: Allegra X-15R ; bench top centrifuge with SX4750 rotor)
   Note: Mtb cultures should be handled in BSL3 facility according to institutional standards of practice.
   
8. 37 °C shaker incubator with aerosol containment units for Mtb liquid cultures
   
9. Beckman aerosolve canisters for centrifuging mycobacterial cultures in falcon tubes (e.g. Beckman Coulter, catalog number: BK359232 )
   
10. Multiwell-Plate Carrier Covers (e.g. Beckman Coulter, more on this link https://www.beckmancoulter.com/wsrportal/techdocs?docname=GX-TB-012)
    
11. 37 °C shaker incubator with aerosol containment units for Mtb liquid cultures
    
12. Epifluorescence microscope [e.g. Nikon Eclipse TiE/B model equipped with 60x; Plan-Apochromat, NA 1.4 oil immersion objective, Ti Z drive, high resolution monochrome charge-coupled device (CCD) digital camera; Photometric Cool SNAP HQ2 and appropriate filter sets for DAPI, FITC and TexasRed channel]
    

Software

1. Nikon Imaging Software-Elements Advanced Research (NIS-Elements AR) version 3.2 software with deconvolution module
   
2. Graph Pad Prism software
   

Procedure