Phagocytosis Assay of Microglia for Dead Neurons in Primary Rat Brain Cell Cultures

Materials and Reagents

1. Cell Strainer (100 μm and 40 μm) (BD, Falcon, catalog number: 431752 and 431750)
   Note: Currently, it is “Corning, Falcon, catalog number: 431752 and 431750 ”.
   
2. Cell Lifter (VWR International, catalog number: 89030-910 )
   
3. 100 mm Petri dish (Thermo Fisher Scientific, catalog number: 263991 )
   
4. 60 mm TC dish (Corning, catalog number: CLS430166 )
   
5. 75 cm² TC flask (Corning, catalog number: 430825 )
   
6. 24-well TC plate (BD, Falcon, catalog number: 353047)
   Note: Currently, it is “Corning, Falcon, catalog number: 353047 ”.
   
7. 1 ml Syringe (BD, Falcon, catalog number: 305217 )
   
8. Glass Pasteur pipet (VWR International, catalog number: 14673-043 )
   
9. E-18 Pregnant Sprague Dawley (SD) rat (Charles River Laboratories International)
   
10. Pentobarbital (Sigma-Aldrich, catalog number: P-3761 )
    
11. Neurobasal medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 21103 )
    
12. B27 Supplement (Thermo Fisher Scientific, Gibco^TM, catalog number: 17504 )
    
13. Glutamine (Sigma-Aldrich, catalog number: G7513 )
    
14. Penicillin/Streptomycin (100x) (GE Healthcare, Hyclone^TM, catalog number: SV30079 )
    
15. DMEM (Corning, catalog number: 10-013-CM )
    
16. Propidium Iodide (PI) (Sigma-Aldrich, catalog number: P-4170 )
    
17. DAPI (4’, 6-Diamidino-2-Phenylindole, Dilactate) (Invitrogen, catalog number: D3571 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, catalog number: D3571”.
    
18. 16% Paraformaldehyde (PFA) (Electron Microscopy Sciences, catalog number: 15170 )
    
19. Fetal Bovine Serum (FBS) (GE Healthcare, Hyclone^TM, catalog number: SH30071 )
    
20. Sodium pyruvate (Sigma-Aldrich, catalog number: P5280 )
    
21. HEPES Buffer solution (Sigma-Aldrich, catalog number: 83264 )
    
22. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
    
23. Poly-D-Lysine (Sigma-Aldrich, catalog number: P0889 )
    
24. HBSS (Lonza, catalog number: 10-543F )
    
25. PBS (Corning, catalog number: 21-040-CV )
    
26. 0.4% Trypan blue (Sigma-Aldrich, catalog number: T8154 )
    
27. Alexa Fluor 488-Phalloidin (Invitrogen, catalog number: A12379 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, catalog number: A12379”.
    
28. Mouse anti-MAP2 antibody (Sigma-Aldrich, catalog number: M4403 )
    
29. Rabbit anti-mouse IgG-Alexa Fluor 488 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A11029 )
    
30. Dissection buffer (see Recipes)
    
31. DMEM/FBS (see Recipes)
    
32. Neurobasal/B27 (see Recipes)
    Notes:
    1. All reagents and chemicals for cell cultures must be sterile. Without special indication, all culture medium or buffer is used at room temperature (RT). And all operational procedures for neuronal culture and microglia (MΦ) culture, including animal dissecting, tissue triturating, cell plating or harvesting, and medium changing, have to be performed in a dissecting hood or cell culture hood using sterile tools.
    2. Poly-D-Lysine coated tissue culture (TC) plate. The TC dish and plate for the primary neuronal cells or purified microglia must be pre-coated with Poly-D-lysine (0.1 mg/ml in H₂O) (2 ml for 60 mm² TC dish, 0.5 ml per well for 24w-TC plate) for 30 min at 37 °C. After washing with sterile water, the TC dishes/plates have to be air-dried in the cell culture hood before use.
    3. The age of the embryos for cell culture. Although E16 embryos are more suitable for neuronal culture and post-natal 1-2 -day-old pups are more suitable for glia culture, we used the E-18 embryos to prepare the neuron culture and also the glial culture (to isolate MΦ). Thus, the neurons and MΦ are prepared from the same litter of embryos. The neuronal culture and microglia can be prepared from embryos or pups of different litters.
    4. The purity of neurons in the cortical neuronal culture. The Neurobasal/B27 medium is serum-free medium, which is optional for neuronal culture. However, there are other cell types in the cultures. In the 2-day-old neuronal cultures, the ratio of MAP2⁺-neurons is 43.6 ± 23% (n = 10, Figure 1).
       
       
       Figure 1. MAP2 immunofluorescence of 2-day-old rat cortical neurons in culture. The cortical neuron culture is fixed in 2% PFA and labeled by mouse anti-MAP2 antibody. The signals are visualized with Alexa Fluor 488 (Green). The nuclei of all cells are labeled with DAPI (red). The arrows indicate the MAP2⁺-neurons. Scale bar = 50 μm.
       
       
    5. All animal studies followed the guidelines outlined in Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and were approved by the Animal Welfare Committee of The University of Texas Health Science Center at Houston.
    6. The Irradiation operation followed the guidelines for Environment Health & Safety of The University of Texas Health Science Center at Houston.
       

Equipment

1. Hemacytometer (VWR International, catalog number: 15170)
   
2. Cell culture CO₂ incubator (LabX, Sanyo, model: MCO-19AIC )
   
3. Fluorescence microscopy with Imaging System, Olympus IX81 controlled by MetaMorph 7.4
   
4. Dissecting microscope (Nikon Corporation, model: SMZ-27 )
   
5. Digital controlled orbital shaker (Thermo Fisher Scientific, model: MaxQ^TM 2000 )
   
6. ¹³⁷Cs Irradiation Source (J. L. SHEPHERD & ASSOCIATES, model: 143 ) (Figure 2)
   
   
   Figure 2. Photos of the ¹³⁷Cs irradiator. The cultured neurons grown in the 60 mm TC dishes are loaded into the metal cylinder at top of the irradiator. Upon pressing the red button on the right controller, the cylinder will rotate and go down until completely being imbedded in the irradiator (the round bottom portion) for irradiation. By the irradiation time (the left controller) is over, the irradiator will automatically rotate the cylinder upward to its original position. By now, the irradiated cultures are ready to be removed from the cylinder and returned to the CO₂ incubator.
   

Software

1. MetaMorph software, version 7.4
   

Procedure