Staining Cell-Surface Antigens With Antibodies (Optional); Cell Fixation And Permeabilization - PromoKine PK-CA724-488HTS Instruction Manual

5.2 For the desired final concentration, add the appropriate amount of EdU to the
culture medium and mix well. We recommend using a concentration of 10 µM
for 1-4 hours as a starting point. Use higher EdU concentrations for a shorter
incubation time. A longer incubation time requires lower EdU concentrations.
5.3 The incubation of the cells with EdU should be performed under the optimal
conditions for your cell type, the number of cells plated and for the desired
length of time. Various DNA synthesis and proliferation parameters can be
evaluated by altering the EdU incubation time or by subjecting the cells to pulse
labeling with EdU. Effective time intervals for pulse labeling and the length of
each pulse depend on the cell growth rate.
5.4 If performing antibody surface labeling, proceed immediately to step 6,
otherwise continue to step 7.

6. Staining Cell-surface Antigens with Antibodies (optional)

6.1 Wash cells in each well with 100 µL of 1% BSA in PBS.
6.2 Remove the wash solution and add again 100µL of 1% BSA in PBS to the cells.
6.3 Add surface antibodies and mix well but gently.
Note: PE, PE-tandem or Quantum Dot antibody conjugates should not be used
before performing the click reaction (step 8).
6.5 Incubate the cells for the recommended length of time and temperature. Protect
from light!
6.6 Proceed to step 7.

7. Cell Fixation and Permeabilization

This protocol was developed with a fixation step using 4% Paraformaldehyde in PBS,
followed by permeabilization step. A saponin-based permeabilization solution can be
used with cell samples containing red blood cells or whole blood as well as with cell
probes containing different cell types. The morphological light scatter characteristics of
leukocytes are maintained by a saponin-bases solution while red blood cells are lysed.
7.1 Remove the incubation media and wash the cells, each well with 100 µL of 1%
BSA in PBS. Afterwards remove the wash solution.
7.2 Add 100 µL of the fixative solution to the cells in each well. Incubate for 15
minutes at room temperature. Protect from light.
7.3 Remove the fixation solution and wash the cells in each well twice with 200 µL
of 1% BSA in PBS. If red blood cells or haemoglobin are present in the sample,
repeat the washing step. Remove all residual blood cell debris and haemoglobin
before proceeding.
NOTE: At this point of the procedure the probes can be stored safely.
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Instruction Manual