Mitochondrial Antigen Presentation in RAW macrophages

Prepare a 24 well plate:

• Label appropriately (experimental condition – Heath shock (HS), controls, infection controls, etc. )
• Duplicate labelled wells for the peptide control

Scrape RAW cells and resuspend (pipette up and down 8x).

Count RAW cells and adjust concentration to 1 million cells/ml .

Add 1mL to each well.

Check if you have good 2E2 hybridoma cells and passage them if necessary.

Incubate at 37°C.

Treatments (Prior to beginning, check the cell’s media red/orange = good, yellow = bad).

Heath shock treatment: incubate the plate in the water bath at 42°C for 0h 45m 0s1. Place the plate at 37°C for 4h 0m 0s

1. 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 250µL and 3 wells of 50µL for peptide
2. Incubate the cells at 37°C for 3h 0m 0s

LPS treatment: 1.5µL (dilute 20µL of LPS stock in 666µL of media, then add 10µL per 1mL of media)1. Place the plate at 37°C for 3h 0m 0s

1. 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 250µL and 3 wells of 50µL for peptide
2. Incubate the cells at 37°C for 3h 0m 0s

Bacteria treatment: bacterial infection is MOI 1. Usually 3µL in 1mL then add 100µL 1. Place the plate at 37°C for 3h 0m 0s

1. 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 250µL and 3 wells of 50µL for peptide
2. Incubate the cells at 37°C for 3h 0m 0s

Prepare 1% (v/v) at 37Room temperature (7.4).

Discard supernatant in the 96 well plate and add 50µL.

Incubate at 37Room temperature for 0h 10m 0s.

Prepare 2E2 cells while RAW cells are in 1% PFA:

Pour flask into 50 ml conical and centrifuge at 1500rpm.

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 250µL of media and the other tube for the peptide control wells with 50µL of media)

Add 200µL 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.

Repeat 2x more: Add 200µL to each well in the 96 well plate and discard immediately. (Wash 1/2)

Repeat 1x more: Add 200µL to each well in the 96 well plate and discard immediately. (Wash 2/2)

Add 250µL per well in ½ of the samples (the non-peptide ones). Add an extra row of 2E2 cells alone as a negative control.

Dilute stock peptide (1 µg/ml) 5000 fold in the 2E2 cells (final concentration: 0.2nanogram per milliliter (ng/mL)).

Add 250µL to the remaining wells. Add an extra row of 2E2 cells+peptide alone as a negative control.

Incubate at 37°C for 16h 0m 0s.

Prepare 1x lysis buffer (stock = 5x, with triton, 7.8) in dH₂O.

Add 30µL in 10mL.

Prepare CPRG solution (7.8).

• 150µL
• 20.2µL
• 0.046mg

Centrifuge 96 well plate at 2200rpm.

Add 50µL to each well.

When lysis is done, add 170µL / well.

Incubate either at 37Room temperature or 37°C for 0h 30m 0s until the sample turns dark red (37°C speeds up reaction to about ~ 20 minutes for peptide samples).

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

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