Phagocytosis

VIEW PRODUCT LISTING ASK A QUESTION

Materials

  • Vial of Monocytes (catalog # 1008 or 1009)
  • Alexa Fluor 488 Phalloidin (or other equivalent)
  • Alexa Fluor 594 Labeled Zymosan (or other equivalent)
  • Coverslips
  • PBS (Phosphate Buffered Saline)
  • Paraformaldehyde

Procedure

  1. Thaw monocytes and suspend in medium at a concentration of 1 million per mL.
  2. Place coverslips into a petri dish or other suitable container and pipet 250–500 microliters of the cells onto the coverslip. Surface tension should keep the fluid on the coverslip.
  3. Prepare zymosan according to manufacturer’s recommendations and count particles to determine concentration.
  4. Add zymosan to the cells at a 10:1 ratio, 10 yeast to each monocyte.
  5. Incubate at 37°C for one hour. Incubating a coverslip at 2–8°C will provide a negative control.
  6. Rinse the coverslips gently with 3–4 exchanges of PBS.
  7. Fix the cells by adding 2% paraformaldehyde to cover the cells and incubate 10 minutes at ambient temperature.
  8. To counterstain, add 0.1% Triton X-100 to cover the cells for 5 minutes.
  9. Rinse three times with PBS.
  10. Add 200 microliters of diluted Alexa Fluor 488 phalloidin.
  11. Incubate 20 minutes at ambient temperature.
  12. Wash three times with PBS.
  13. Place a small drop of 50% glycerol or other mounting medium on a microscope slide and invert the coverslip, cell side down, onto the slide.
  14. Examine under a fluorescent microscope.