Macrophage Survival Assay Using High Content Microscopy

Materials and Reagents

1. Sterile 1.5 ml tubes (Corning, Axygen^®, catalog number: MCT-175-C )
   
2. 25 G-needle
   
3. 50 ml syringe (B. Braun Medical, catalog number: 4617509F-02 )
   
4. Polypropylene conical 50 ml centrifuge tube (TPP Techno Plastic Products, catalog number: 91050 )
   
5. 40 µM cell strainer (Corning, Falcon^®, catalog number: 431750 )
   
6. 90 mm Petri dish (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 101RTC )
   
7. 96-well clear bottom cell-culture grade black imaging plates (Corning, Falcon^®, catalog number: 353219 )
   
8. Sterile reagent reservoir (VWR, catalog number: 89094-664 )
   
9. 10 ml serological pipette (SARSTEDT, catalog number: 86.1254.001 )
   
10. 25 ml serological pipette (SARSTEDT, catalog number: 86.1685.001 )
    
11. 10 μl filtered barrier tip (Biotix, Neptune^®, catalog number: BT10XL )
    
12. 200 μl filtered tip low retention (CLEARLINE, catalog number: 713117 )
    
13. 0.22 μm syringe-filter (Carl Roth, catalog number: P668.1 )
    
14. Adhesive plate seal
    
15. 6-to-9 week old specific-pathogen free C57BL/6 mice
    
16. ddH₂O
    
17. Ethanol
    
18. Macrophage colony stimulating factor (M-CSF) (ImmunoTools, catalog number: 12343115 )
    
19. EDTA 0.5 M pH 8.0 solution (as described Reference 1)
    
20. Pharmacological inhibitor
    
21. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270106 )
    
22. HEPES buffer (BioConcept, catalog number: 5-31F00-H )
    
23. Penicillin-streptomycin (P/S) (BioConcept, catalog number: 4-01F00-H )
    
24. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 31966021 )
    
25. Sodium hydroxide (NaOH)
    
26. Hydrochloric acid (HCl)
    
27. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 76240 )
    Note: This product has been discontinued.
    
28. Cell-culture grade Ca/Mg-free Dulbecco’s PBS (DPBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14040091 )
    
29. Saponin (Sigma-Aldrich, catalog number: 84510 )
    
30. DAPI (Thermo Fisher Scientific, Invitrogen^TM, catalog number: D1306 )
    
31. Phalloidin (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A12379 )
    
32. Complete DMEM cell medium (see Recipes)
    
33. 4% PFA solution (see Recipes)
    
34. 5% Saponin solution (see Recipes)
    
35. Staining solution (see Recipes)
    

Equipment

1. 10-11 cm long stainless steel dissecting scissors
   
2. 10-11 cm long stainless steel dissecting straight forceps
   
3. Refrigerator centrifuge with 50 ml tube adapter
   
4. Cell culture incubator
   
5. Pipette controller
   
6. Pipettes
   
7. Cell counting chamber
   
8. Finnpipette^® 50-300 μl 12 channel multi-pipette
   
9. Chemical hood
   
10. Luminal flow hood
    
11. Plate washer (BioTek Instruments, model: EL406 )
    
12. High content microscope (such as Molecular Devices, model: ImageXpress Micro XL )
    
13. 40x Plan Apo λ 0.95 NA objective (Nikon, catalog number: MRD00405 )
    
14. pH meter
    

Procedure