Lab Experiment 33.2: Study the interaction between phagosome and lysosone in J774A.1 macrophage cells.
Material and Instrument
1. Methanol
2. Acetone
3. PBS (1X)
4. 1% Triton X-100
5. BSA (Fat free, acetylated): Prepare 5% BSA solution in PBS and filter with the 0.45mm filter to rmove particulate matter.
6. Primary antibody (anti-protein): An antibody can be developed against protein (antigen of interest) in rabbit or mice.
7. Secondary antibody: An antibody coupled with fluorescent marker (such as FITC) and directed against mouse IgG.
8. Epi-fluorescence microscope
9. 1µm Latex Beads
10. Filipin: Prepare 5mg/ml stock solution of filipin in 100% alchol. The working solution is 50µg/ml in PBS. 
11. Rhodamine Dextran.

Procedure:

A. Identification of phagosome:
1. J774A.1 cells are cultured in the DMEM media containing 10% FBS and 1% antibiotics cocktails (pencillin/streptomycin sulphate).
2. Remove the cells from the cell culture plate by trypsinization or by 0.5% EDTA in PBS.
3. Plate 10,000 cells on 12mm cover glasses and incubate it in the 24 well dish with 0.5ml DMEM media containing FBS and antibiotic cocktail.
4. Incubate cells over night at 37⁰C and 5% CO₂ and it will allow the cells to attach to the cover glasses. 
5. Wash the cells with DMEM without FBS media.
6. Prepare a suspension of latex beads (10⁶ beads/ml) in DMEM without FBS media.
7. Remove media and add beads suspension to the well and centrifuge the 24 well dish at 1000rpm for 1mins at 4⁰C.
8. Incubate the plate for 1hrs at 37⁰C and 5% CO₂.
9. Wash the well with 1ml DMEM without FBS media to remove unentrenalized beads.
10. Fix the biological sample with Methanol: Acetone (7:3) mixture at -20⁰C for 15 min. Hydrate the sample with 1X PBS.
11. Stain the cells with filipin (50µg/ml) for 1hrs at 370C in dark.
12. Keep one drop (~20µl) of mounting medium (glycerol mounting media containing antifading agent) on the glass slide and keep the cover glass on it. Firm the cover glass by making a thick rim by nail polish.