Staining protocol for Gerard – 8/10/2014


Original Author: Orsolya Symmons

Modified by: Gerard Triqueneaux


Aim of experiment:

  1. validate the results/expression differences for CD38 between the cell lines

  2. test if DAPI staining is compatible with ImageStream (can be used for cell cycle differentiation)

  3. test new fixation conditions

For the antibodies that worked together with the DAPI+20min protocol (CD19, CD22, CD38), do staining on all cell lines. 4 tubes/cell line: 1 with only DAPI, 1 each for all the single stainings.

  • Prepare tubes for staining, take out PFA to equilibrate to RT. Prepare 1 tube with PBS at RT.

  • Count cells. Take x cells per cell line.

NB1: All my experiments were done using 0.5 x106 cells/staining, so all quantities are given for this number of cells – please scale accordingly.

NB2: I generally run a negative (unstained) control for each cell line, but if that is too much for you then you can mix the cell lines and have one “combined control”

  • spin cells @4ºC, 400g 5min

  • meanwhile prepare master mix of antibodies (100µl FACS buffer+2.5µl CD38 PE/Cy7 antibody -> MM: 650 µl FACS buffer+16.25µl antibody)

  • remove medium. Resuspend each sample in 200µl ice-cold FACS buffer.

  • Split each tube into 2x 100µl. To one tube add 100µl FACS buffer, to the other tube add 100µl of antibody

  • Put in cold-room 30min, rotating

  • spin cells @ 400g, 4ºC 5min

  • resuspend cells in 500ml FACS buffer

  • spin cells @ 400g, 4ºC, 3 min -> make sure to keep lid of centrifuge open, so it can equilibrate to RT.

  • to each tube add 400µl 4% PFA

  • keep at RT 20'

  • after fixation, spin cells @ 400g, RT, 5min

  • wash with 500µl PBS

  • spin cells @ 400g, RT, 3min – split each tube into 2 tubes

  • resuspend 1 tube cells in DAPI solution and 1 tube in PBS

FACS buffer: PBS+10% FCS+150µl 0.1M NaN3/10ml solution -> ie for 10ml FACS buffer: 9ml PCS+1ml FCS+150µl NaN3

DAPI solution: PBS+10% FCS+1.25µl DAPI/ml -> ie for 10ml DAPI solution:
9ml PBS+1ml FCS+12.5µl DAPI