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Basic Antibody Staining

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Basic Antibody Staining

This protocol is for cultured cells.

For each monoclonal antibody (MAb) marker use 1 X 105 - 1 X 107 cells per 100 µl volume:

  1. If adherent cells, trypsinize, scrape or treat with EDTA to get single cells suspension, otherwise skip to step 2.
  2. Wash cells with 2 ml COLD Phosphate Buffered Saline (PBS) or another buffer with 0.1%-5% protein (eg Bovine Serum Albumin, BSA) by centrifuging at 150-300 X g in the COLD (4°C).
  3. Wash once more (Optional).

Staining:

  1. Aliquot 100 µl of 1-100 X 105 cells (use wash buffer above with 0.1% NaN3 added) in FalconŽ #2052 or #2054 tubes on ice.
  2. Add 20 µl MAb (1-10 µg/ml final concentration, depending on Mab).
  3. Incubate 10 - 30 min on ice.
  4. Wash again as above with 2 ml cold azide buffer.
  5. Resuspend in 100 µl of Secondary Antibody (eg. Fluorescinated Goat anti-mouse IgG). Otherwise skip to step 7 if using directly conjugated MAb.
  6. Incubate 10 - 30 min on ice in DARK.
    Wash again with azide buffer.
  7. Resuspend in 100 to 500 µl COLD wash buffer and add equal volume of COLD buffered 2% para-formaldehyde (see recipe) to fix cells
    OR leave in wash buffer and analyze LIVE (Add propidium iodide, 0.5 µg/ml final concentration).
    Ideal final concentration of cells should be 1 X 106/ml.

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Dennis J. Young, UCSD Cancer Center Flow Cytometry Core Facility

 


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