TITLE: Mitochondrial Antigen Presentation in RAW macrophages
AUTHORS: Ahmed Fahmy, Tyler Cannon

[DESCRIPTION]
This protocol details methods for 3-day Mitochondrial Antigen Presentation Assay in a murine macrophage cell line (RAW) that expresses a glycoprotein B (gB) of herpes simplex virus 1 (HSV1) targeted to the mitochondrial matrix (mito-gB). A gB-specific CD8+ T cell hybridoma recognizing the gB498–505peptide loaded on MHC class I molecules is also used to monitor antigen presentation through a beta-galactosidase assay kit.

[STEPS]
SECTION: Day 1: cell plating
1. Prepare a 24 well plate:
Label appropriately (experimental condition – Heath shock (HS), controls, infection controls, etc. ) 
Duplicate labelled wells for the peptide control
SECTION: Day 1: cell plating
2. Scrape RAW cells and resuspend (pipette up and down 8x).
SECTION: Day 1: cell plating
3. Count RAW cells and adjust concentration to 1 million cells/ml.
SECTION: Day 1: cell plating
4. Add 1 mL to each well.

SECTION: Day 1: cell plating
5. Check if you have good 2E2 hybridoma cells and passage them if necessary. 
SECTION: Day 1: cell plating
6. Incubate at 37 °C. 

SECTION: Day 2: Treatment, fixation and hybridoma incubation
7. Treatments (Prior to beginning, check the cell’s media red/orange = good, yellow = bad).
SECTION: Day 2: Treatment, fixation and hybridoma incubation
8. Prepare 1 % (v/v) at 37 Room temperature (pH 7.4).

SECTION: Day 2: Treatment, fixation and hybridoma incubation
9. Discard supernatant in the 96 well plate and add 50 µL. 

SECTION: Day 2: Treatment, fixation and hybridoma incubation
9.1. Incubate at 37 Room temperature for 10 min.

SECTION: Day 2: Treatment, fixation and hybridoma incubation
10. Prepare 2E2 cells while RAW cells are in 1% PFA:
SECTION: Day 2: Treatment, fixation and hybridoma incubation
10.1. Pour flask into 50 ml conical and centrifuge at 1500 rpm, 3 min. 
 

SECTION: Day 2: Treatment, fixation and hybridoma incubation
10.2.  Count 2E2 cells and adjust concentration to 0.4 million cells/ml. Make enough for all the wells and split volume in 2 tubes (you will need 1 tube to add to cells in 250 µL of media and the other tube for the peptide control wells with 50 µL of media)

SECTION: Day 2: Treatment, fixation and hybridoma incubation
11. Add 200 µL to each well in the 96 well plate and discard immediately.
! Make sure to wash the cells gently so as to not lose them. Add buffer against the wall of the well.
 


SECTION: Day 2: Treatment, fixation and hybridoma incubation
11.1. Repeat 2x more: Add 200 µL to each well in the 96 well plate and discard immediately. (Wash 1/2)
SECTION: Day 2: Treatment, fixation and hybridoma incubation
11.2. Repeat 1x more: Add 200 µL to each well in the 96 well plate and discard immediately. (Wash 2/2)
SECTION: Day 2: Treatment, fixation and hybridoma incubation
12. Add 250 µL per well in ½ of the samples (the non-peptide ones). Add an extra row of 2E2 cells alone as a negative control. 

SECTION: Day 2: Treatment, fixation and hybridoma incubation
13. Dilute stock peptide (1 µg/ml) 5000 fold in the 2E2 cells (final concentration: 0.2 nanogram per milliliter (ng/mL)).
SECTION: Day 2: Treatment, fixation and hybridoma incubation
14. Add 250 µL to the remaining wells. Add an extra row of 2E2 cells+peptide alone as a negative control.

SECTION: Day 2: Treatment, fixation and hybridoma incubation
15. Incubate at 37 °C for 960 min.
SECTION: Day 3: Lysis and revealing
16. Prepare 1x lysis buffer (stock = 5x, with triton, pH 7.8) in dH2O.

SECTION: Day 3: Lysis and revealing
16.1. Add 30 µL in 10 mL.

SECTION: Day 3: Lysis and revealing
17. Prepare CPRG solution (pH 7.8).
150 µL 
20.2 µL 
0.046 mg 


SECTION: Day 3: Lysis and revealing
18. Centrifuge 96 well plate at 2200 rpm, 1 min.

19. Add 50 µL to each well.

20. When lysis is done, add 170 µL / well. 

20.1. Incubate either at 37 Room temperature or 37°C for 30 min until the sample turns dark red (37°C speeds up reaction to about ~ 20 minutes for peptide samples).

21. Transfer 150 µL to a new plate. Stop the reactions by adding 80 µL  of stop solution to each well
 



22. Read in a plate reader at a wavelength of 570–595 nm.
 

SECTION: Day 2: Treatment, fixation and hybridoma incubation
7.1. Heath shock treatment: incubate the plate in the water bath at 42 °C for 45 min
Place the plate at  37 °C for 240 min 
Take the cells and re-plate in a 96 well plate. 1 well of a 24 well plate will be distributed in 6 wells = 3 wells of 250 µL and 3 wells of 50 µL for peptide
Incubate the cells at 37 °C for 180 min 

SECTION: Day 2: Treatment, fixation and hybridoma incubation
7.2. LPS treatment: 1.5 µL (dilute 20 µL of LPS stock in 666 µL of media, then add 10 µL per 1 mL of media)
Place the plate at 37 °C for 180 min 
Take the cells and re-plate in a 96 well plate. 1 well of a 24 well plate will be distributed in 6 wells = 3 wells of 250 µL and 3 wells of 50 µL for peptide
Incubate the cells at 37 °C for 180 min 

SECTION: Day 2: Treatment, fixation and hybridoma incubation
7.3. Bacteria treatment: bacterial infection is MOI 1. Usually 3 µL in 1 mL then add 100 µL 
 Place the plate at 37 °C for 180 min 
Take the cells and re-plate in a 96 well plate. 1 well of a 24 well plate will be distributed in 6 wells = 3 wells of 250 µL and 3 wells of 50 µL for peptide
Incubate the cells at 37 °C for 180 min