Cell surface stability measurement by 1Ab labeling

Materials:

- Ice-cold PBS+1mM MgCl2+ 0.1mM CaCl2
- Ice-cold phenol-free growth medium supplemented
- 4% paraformaldehyde in PBS
- Primary (1Ab) and Secondary (2Ab) antibodies
- AmplexRed assay reagents


Cells preparation
Day before assay split cells in twelve well plates as triplicates or quadruplet samples and grow them till cell will reach 80-90% confluence. Always keep additional wells for secondary 2Ab background control.

Experiment procedure:

1. Wash each well on ice with ice-cold PBS+1mM MgCl2+ 0.1mM CaCl2. Repeat the step above.
2. 30 min. Incubation on Ice, with 2.5% FCS Ice-cold (blocking)
2. Incubate cells with primary 1Ab in phenol-free growth medium supplemented with 5%FCS for 1hour on ice.
3. Wash three times with ice-cold PBS+1mM MgCl2+ 0.1mM CaCl2 .


At that stage cells can be kept at 40C, for additional time point samples can be fixed with 4% PFA .

4. Incubate cells with secondery Ab in phenol-free growth medium supplemented with 5%FCS for 1hour on ice.
5. Wash with ice-cold PBS+1mM MgCl2+ 0.1mM CaCl2, five times and detect the cell surface stability by AmplexRed assay of HRP activity