Nov 01, 2019
Cell Surface Mild Acid Elution of MHC-bound Immunopeptides
- 1University of British Columbia
- Leonard Foster's Lab
Protocol Citation: Teesha C Luehr 2019. Cell Surface Mild Acid Elution of MHC-bound Immunopeptides. protocols.io https://dx.doi.org/10.17504/protocols.io.y6ffzbn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 13, 2019
Last Modified: November 01, 2019
Protocol Integer ID: 21415
Culturing Cells
Culturing Cells
Culture cells. If culturing THP-1s, follow the protocol.
The base medium for this cell line is RMPI-1640
Reguired supplements:
- 1 % volume L-glutamine
- 10 % volume Fetal Bovine Serum
Note
Most catalog numbers of RMPI-1640 contain L-glutamine, however, some do not. Ensure that it is in the media before using it for culturing.
Optional Supplements:
- 1 % volume PenStrep
- 0.05 millimolar (mM) 2-mercaptoethanol
Note
PenStrep is not required for THP-1 culturing, however, if you are having issues with bacterial contamination, it can be used at 1X.
Note
2-mercaptoethanol is stated as a required component for complete RPMI-1640 medium, however, in our laboratory it is not standard practice to add it.
Always store cells in liquid nitrogen. This is for both the original tube of cells from ATCC and any passages afterwards.
Place the media bottle in the 37 °C water bath at least 00:30:00 prior to using
Thaw cells at 25 °C (room temperature) for 00:10:00 or 37 °C in a water bath for 00:02:00
Sanitize all items going into the Biological Safety Cabinet with 70% ethanol
As soon as the cells are thawed, transfer the cells to a 15 mL conical tube and add 10 mL of complete media
Note
Cells are stored with 5% DMSO, which can lyse cells if they are left for too long.
Pellet cells for 00:03:00 at 500g
Discard supernatant
Resuspend cells by pipetting up and down 5X in 5 mL complete media
Transfer cells + media to a T-25 flask
Incubate cells at 37 °C and 5% CO2 and 80% humidity
THP-1 cells replicate after ~26 hours. In practice, it takes 2 days for a true doubling.
Once cells have doubled OR when media has begun to change colour, it is time to add media, split cells into new flasks, or to spin down to remove all media
Adding media4 steps
If concerned about cell concentration, perform a cell count
Double the total media volume with new complete media
Carefully mix the new media in by rocking the flask back and forth
Place the flask back in the incubator
Grow cells to a total number of 1-2x10*8 cells
Preparing Reagents
Preparing Reagents
1X PBS at 25 °C (room temperature)
- 8 g NaCl
- 0.2 g KCl
- 1.44 g NA2HPO4
- 0.24 g KH2PO4
- 1 L Milli-Q water
- pH adjust to 7.4 with 37% HCl
1X PBS at 4 °C
Note
This PBS solution does NOT have to be sterile, but make sure it is clearly marked.
1X saline (PBS without the phosphate) at 4 °C
- 4 g NaCl
- 0.1 g KCl
- 500 mL Milli-Q water
- no need to pH adjust, it's not a buffer
- no need to sterilize as the mild acid elution is not done under sterile conditions
1X saline + 2% acetic acid at 4 °C
- 245 mL of above 1X saline solution
- 5 mL acetic acid
Each 250 mL T-175 flask will require 7 50 mL conical tubes
Pelleting & Rinsing Cells
Pelleting & Rinsing Cells
Cells will most likely be in 250 mL complete media . Use five 50 mL conical tubes to pellet all cells from one flask at a time.
Pellet cells for 00:03:00 at 500g
Discard supernatant
Resuspend each pellet by pipetting up and down 5X with 5 mL room temp PBS (sterile in the hood)
Combine all five conical tubes into one
If cell count is not know, perform one now to confrim a minimum of 1x10*8 cells
Pellet cells for 00:03:00 at 500g
Discard supernatant
Resuspend each pellet by pipetting up and down 5X with 10 mL cold PBS
Pellet cells for 00:03:00 at 500g
Discard supernatant
Resuspend each pellet by pipetting up and down 5X with 10 mL cold PBS
Pellet cells for 00:03:00 at 500g
Discard supernatant
Resuspend each pellet by pipetting up and down 5X with 10 mL cold saline
Transfer cells + saline to a new 50 mL conical tube
Note
This is to remove any large amounts of phosphate
Pellet cells for 00:03:00 at 500g
Discard supernatant
Mild Acid Elution
Mild Acid Elution
Resuspend each pellet by pipetting up and down 5X with 10 mL cold 2% acetic acid in saline
Note
EXTREMELY IMPORTANT, REMEMBER THAT THE PEPTIDES WILL BE IN THE SUPERNATANT
DO NOT DISCARD SUPERNATANT
Collect the supernatant in a new 50 mL conical tube
Drying
Drying
Freeze supernatant at -80 °C overnight
Lyophilize sample for 48 hours
Peptides are now ready to be desalted with chosen clean up method (STAGE tip, LC-UV, combination, etc)