Survival or Killing Assay Protocol for Autophagy

Survival or Killing Assay Protocol for Autophagy (For working with 6 strains of Mycobacteria)

1. Subculture Raw 264.7 into two 75 cm² tissue culture flask (1:10) and incubate for 2 day.
2. One day before the experiment, remove old media and add 10 ml complete DMEM into each flask.
3. Scraps off the cells into 10 ml complete DMEM and pipet up and down several times to get single cell suspension.
4. Then merge the media of two flasks into one flask and pipet the media up and down several times to get a homogenous cell suspension so that it can be used as a single batch and 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 (total 60 ml for working with 6 strains of Mtb). Cells suspension is diluted so that they can grow without any nutritional stress in well plates. Because, any stress can induce autophagy. Moreover, the known number of cells per ml will be needed to calculate the Multiplicity Of Infection (MOI) for infecting with Mtb.
7. Label the 12-well plates according to strains of Mtb and experimental condition. Triplicate for each experimental condition. Dispense 1ml diluted cell suspension into each well of a 12-well plate using 10 mL pipets.  Avoid any air bubble in tip of the pipette during pipetting.
8. Incubate the well-plate at 37⁰ C, 5% CO₂ overnight. Here, 37⁰ C is used because mammalian cells require this temperature to grow. 5% CO₂ is used in order to maintain the pH to be at 7.4
9. After incubation, carry the 12 well plates into BSL-3.

In BSL-3

10. Keep the plates as soon as possible at 37⁰ C, 5% CO₂ in ⁱncubator _in BSL-3
11. Turn on OD₆₀⁰ machine and Centrifuge machine to be ready them.
12. Put DMEM into water bath to warm it.
13. Prepare safety hood for your immediate work. {Clean the floor of hood with 8% ADBAC, Put a waste container, a waste bottle for liquid waste, a rack or conical flask holder, 6 conical flasks, pipette and pipette gun, Complete DMEM and PBS bottle in hood. For first time entry decontaminate everything with 70% ethanol, But, in case of all out  (1^st out also) from hood everything  must be decontaminated with 8% ADBAC}
14. In hood label 6 conical flasks(50ml) according to Mtb strains.
15. Add 45 ml PBS in each flask. PBS is used here to wash out Tween-80 which will affect the infection.
16. Add 5ml of a log-phase Mtb culture into each conical flask according to label. 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.
17. Centrifuge at 2500 or 3200 rpm for 5 minutes and remove the supernatant. Thus the media used for growth will be washed away.
18. Re-suspend the Mycobacteria pellet in 6 ml complete DMEM.
19. Transfer the suspension to a 7ml Dounce homogenizer and homogenize 35 times to generate single cell suspension.
20. Measure OD₆₀₀ of the 1:10 (900 µL medium + 100 µL suspension) dilution of homogenized culture. First, put 900 µL medium in each cuvet and then measure OD₆₀₀ one by one. 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.
21. Prepare Mycobacterial inoculum in DMEM at the concentration of 3x10⁶ Mtb/ml (MOI=10) using this formula:

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

22. Transfer Raw cells from incubator to Biosafety cabinet. 
23. Remove media from Raw cells and add 1ml of 3x10⁶ Mtb/ml inoculum into each well of Raw 264.7 cells. 
24. Spin at 1200 rmp for 5 min at room temperature to settle Mtb on cells. It will help Raw cell to internalize Mtb.
25. Incubate the plate for 1 hour at 37⁰ C, 5% CO₂ in incubator. Within this time Mtb will be internalized by Raw cells and now be in mycobacterial phagosomes (not early phagosomes) which mimic the condition in human host.
26. After incubation, quickly wash each well three times with 2 ml PBS to remove free Mtb which were not internalized by Raw cells. Any delay may induce autophagy in cells because of stress. Also, this step will remove any residual DMEM which will affect starvation.
27. Add 1 ml DMEM in each well labeled as Full / Add 2 ml EBSS in each well labeled as starvation. Incubate the Full and Starvation plates for 4 hours at 37⁰ C, 5% CO₂ in incubator. Here, starvation media is used for induce autophagy in raw cells. 4 hours incubation period is used because this time is enough for complete autophagic flux to kill Mtb. 
28. After the incubation, wash cells 3 times with 1 mL PBS to remove the media which will affect the plating. Make sure to pipet out all the liquid at the last wash.
29. Add 1 ml of cold deionized water to each well labeled as 0 (zero) hour. For full and starvation plates do this after 4 hours of incubation. Cold deionized water is used for lysis of cell by osmotic burst of cells. Keep it cold to minimize the affect by proteases/lipases that are now release into the water.
30. After adding cold deionized water  incubate the plates for 10-15 minutes at 4⁰C to lyse the cells and release intracellular Mtb by osmotic burst of cells.
31. Prepare dilution plate by adding 180 µL of PBS-Tween into each well of a 96-well plate
32. Pipet the cell lysate up and down to mix and transfer 20µL of mixture into a well of the dilution plate containing 180 µL of PBS-Tween. Thus, make 10^-1, 10^-2, 10^-3 and 10^-4 dilution (10 fold dilution).
33. Inoculate 5 µL of each dilution on 7H11 agar. (To save plates, 12 dilutions can be inoculated onto different slots of a plate)
34. Let the plate stand for 5 minute to absorb the inoculums. Put plates in plastic bag. Close the bag tightly so that plates are prevented from drying out and also from contamination with other organisms. 
35. Incubate the plates at  37⁰ C for 14 days because Mtb grow very slowly. Check after 5 days if there is any colony on the plates, if there is any colony onto plates it is colony of other microbes rather than Mtb because Mtb colony never be visible within 5 days.  
36. After 14 days count colonies from dilution that yield good resolution (1-100 visible colonies). If no visible colony yet let it grow for few days more.
37. Repeat the entire assay two more times.