C1q Binding to and Uptake of Apoptotic Lymphocytes by Human Monocyte-derived Macrophages

Materials and Reagents

1. Human peripheral blood
   
2. RPMI1640 + L-Glutamine + HEPES (Life Technologies, catalog number: 22400-105 )
   
3. FBS (inactivate for 30 min at 56 °C) (Hyclone defined FBS, catalog number: SH30070.03 )
   
4. Penicillin/Streptomycin (Life Technologies, catalog number: 15070-063 )
   
5. L-Glutamine (200 mM) (Life Technologies, catalog number: 25030-081 )
   
6. 0.4% Trypan blue Solution (Sigma-Aldrich, catalog number: T8154 )
   
7. HBSS (Corning/Cellgro, catalog number: MT-21-023-CV )
   
8. PBS
   
9. 25% Human Serum Albumin (HSA) (Plasbumin^®-25) (Talecris Biotherapeutics, NDC number: 13533-684-20 )
   
10. Recombinant human IL-2 (Peprotech, catalog number: 200-02 )
    
11. Recombinant human M-CSF (Peprotech, catalog number: 300-25 )
    
12. Bovine Serum Albumin (BSA) (albumin from bovine serum, lyophilized powder, ≥ 96%) (Sigma-Aldrich, catalog number: A2153-100G )
    
13. C1q (Comptech, Texas, catalog number: A099 )
    
14. PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich, catalog number: PKH26GL )
    
15. CellStripper Dissociation Reagent (Thermo Fisher Scientific, catalog number: 25-056-CI )
    
16. Apoptosis detection kit (BioVision, catalog number: K101-100 )
    
17. Anti-human C1q (Quidel, catalog number: A201 )
    
18. FITC conjugated anti-mouse IgG (Jackson Immunoresearch, catalog number: 115-096-006 )
    
19. FcR blocking reagent human (Miltenyi, catalog number: 130-059-901 )
    
20. FITC conjugated anti-human CD11b (Life Technologies, catalog number: CD11B01 )
    
21. FITC mouse IgG1 isotypes (Life Technologies, catalog number: MG101 )
    
22. Fluorescein phalloidin (Life Technologies, catalog number: F432 )
    
23. Stericup (Thermo Fisher Scientific, catalog number: SCGVU11RE )
    
24. Prolong gold antifade reagent (Life Technologies, catalog number: P36930 )
    
25. Ammonium Chloride (anhydrous)
    
26. Potassium bicarbonate
    
27. Disodium EDTA
    
28. NaN₃
    
29. Trypsin
30. Complete media (see Recipes)
    
31. Phagocytosis buffer (see Recipes)
    
32. ACK buffer (see Recipes)
    
33. FACS buffer (see Recipes)
    

Equipment

1. 50 ml conical tubes (Thermo Fisher Scientific, catalog number: 339653)–important as monocytes stick to other tubes
   
2. 12 x 75 mm polypropylene round bottom sterile tube (Thermo Fisher Scientific, catalog number: 14-956-1D )
   
3. 12 x 75 mm polystyrene round bottom tubes (Thermo Fisher Scientific, catalog number: 14-961-13 )
   
4. Tissue culture plates and vented flasks (any size)
   
5. Eppendorf microfuge tubes
   
6. 12 mm cover slips (Thermo Fisher Scientific, catalog number: GG12 ), sterilize by soaking in 70% Ethanol for 2 x 5 min
   
7. Non tissue culture treated 100 mm petri dish (Thermo Fisher Scientific, catalog number: 0875712 )–referred to as petri dishes
   
8. Tissue culture hood
   
9. Gamma-irradiator
   
10. 5% CO₂, 37 °C Humidified incubator
    
11. Centrifuge for 50 ml conical tubes and 5 ml bottom round tubes
    
12. Centrifuge with swinging bucket rotor for plates (Sorvall, model: RT7000 or equivalent)
    
13. Optic and confocal fluorescence microscopes
    
14. Hemocytometer
    
15. Flow cytometer
    
16. Automatic pipettes (full range volumes)
    
17. Tips (full range volumes)
18. 24-well plates
    

Procedure