Staining protocol for Gerard – 8/10/2014
Original Author: Orsolya Symmons
Modified by: Gerard Triqueneaux
Aim of experiment:
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validate the results/expression differences for CD38 between the cell lines
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test if DAPI staining is compatible with ImageStream (can be used for cell cycle differentiation)
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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.
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Prepare tubes for staining, take out PFA to equilibrate to RT. Prepare 1 tube with PBS at RT.
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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”
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spin cells @4ºC, 400g 5min
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meanwhile prepare master mix of antibodies (100µl FACS buffer+2.5µl CD38 PE/Cy7 antibody -> MM: 650 µl FACS buffer+16.25µl antibody)
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remove medium. Resuspend each sample in 200µl ice-cold FACS buffer.
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Split each tube into 2x 100µl. To one tube add 100µl FACS buffer, to the other tube add 100µl of antibody
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Put in cold-room 30min, rotating
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spin cells @ 400g, 4ºC 5min
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resuspend cells in 500ml FACS buffer
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spin cells @ 400g, 4ºC, 3 min -> make sure to keep lid of centrifuge open, so it can equilibrate to RT.
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to each tube add 400µl 4% PFA
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keep at RT 20'
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after fixation, spin cells @ 400g, RT, 5min
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wash with 500µl PBS
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spin cells @ 400g, RT, 3min – split each tube into 2 tubes
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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