Protocol for Flow cytometry

Flow cytometry

Prepare Cells (day 1)

1. Subculture Raw 264.7 into 75 cm² tissue culture flask (1:10) and incubate for 2 day.
2. One day before the experiment, remove old media and add 5 ml complete DMEM into flask.
3. Scraps off the cells into 5 ml complete DMEM and pipette up and down several times to get single cell suspension.
4. Then pipet the media up and down several times to get a homogenous cell suspension to make cells to be single cells as clumping cells will affect the assays.
5. Determine the cell density using hemocytometer. If possible stain the cell using Tryphan blue before counting with hemocytometer so that the live and dead cell can be distinguished.  Since, cells are very selective in the compounds that pass through the membrane, in a viable cell trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are shown as a distinctive blue colour under a microscope.
6. Dilute cells to 3X10⁵ cells/ml in complete DMEM.
7. Label the 12-well plates according to experimental condition.
8. Dispense 1ml diluted cell suspension into each well of a 12-well plate using 10 mL pipettes.  Avoid any air bubble in tip of the pipette during pipetting.  
9. Incubate the well-plate at 37⁰ C, 5% CO₂ overnight.

Stain Bacteria and prepare inoculum (day 2)

10. In hood add 5ml of a log-phase bacteria culture into 45 ml of PBS in 50 ml conical flask. Log phase culture is used because the least dead cells is present this phase. Make sure that each tube is exactly 50 mL in order to balance the tubes for the centrifugation.
11. Centrifuge at 2500 rpm for 8 minutes and remove the supernatant. Thus the media used for growth will be washed away.
12. Resuspend the pellet in 0.5 ml of 1X PBS by pipetting up down and transfer the cell suspension into a 1.5-ml microcentrifuge tube.  
13. Add 5μl of Alexa 488 stock solution in microcentrifuge tube and mix by pipetting. (Turn of the light of Biosafety cabinet to avoid photobleaching)
14. Wrap the microcentrifuge tube with Aluminum foil. Then, incubate the flask at room temperature for 60 minute on a shaker.
15. Then, pellet bacteria at 8000 rpm for 3 minutes at room temperature. Remove the supernatant and wash twice with1 ml of 1X PBS. Observe the pellet if any green trace is found then wash again.
16. Pipette 5 ml complete DMEM in a 7ml Dounce homogenizer
17. Add 1 ml complete DMEM in each microcentrifuge tube and mix by pipetting. Transfer the suspension to 7ml Dounce homogenizer and homogenize 35 times to generate single cell suspension. 
18. Measure OD₆₀₀ of the 1:10 (900 µL medium + 100 µL suspension) dilution of homogenized culture. First, put 900 µL medium in a cuvet and then measure OD₆₀₀. Each time put 900 µL medium in Machine and set blank for this and then add 100 µL suspension in media, pipet up and down to mix, lid the cuvet with parafilm and finally measure OD₆₀₀ and keep the record.
19. Prepare bacterial inoculum in DMEM at the concentration of 3x10⁶ bacteria/ml (MOI=1:10) using this formula:

(3x10⁶) x (total ml needed for infection / (OD₆₀₀ x 10⁹) = mL homogenate needed.

Phagocytosis

20. Transfer Raw cells from incubator to Biosafety cabinet. 
21. Remove media from Raw cells and add 1ml of 3x10⁶ bacteria/ml inoculum in a well and DMEM medium without bacteria (Control) into anther well. 
22. Spin at 1200 rpm for 5 min at room temperature to settle bacteria on cells. It will help Raw cell to internalize bacteria.
23. Incubate the plate for 30 minutes at 37⁰ C, 5% CO₂ in incubator. Within this time bacteria will be internalized by Raw cells.
24. After incubation, terminate phagocytosis by adding 1 ml of ml of ice-cold PBS in each tube
25. Quickly wash each well two times with 1 ml of ice-cold PBS to remove free bacteria which were not internalized by Raw cells.
26. Then add 1 ml of ice-cold PBS in each well and detach the adherent RAW264.7 cells by GENTLY scrapping well surface using a cell scrapper and transfer the cell suspension from a well to a polypropylene tube. 
27. Centrifuge at 1200 rpm for 5 minute and then remove supernatant
28. Add 500 uL ice cold PBS in each polypropylene tube
29. Keep on ice until flow analysis.