Chemical properties:
PBS+: PBS with 100mg/L of CaCl2 and 100mg/l MaCl2
Dil --MW 933.88, 1mM, CAS number 41085-99-8 (Life Technologies )
NucBlue® Live cell stain ( Life Technologies #R37605)
MSC Attachment Solution (Biological Industries)
1. Cell membrane staining with DiI
1.1 Prepare working solutions: Dilute the stock solution (1mM Dil) into PBS to make 3uM
working solutions.
1.2 Detach cells with trypsinization, and suspend cells at a density of 1 × 106/mL in dye working
solution (from step 1.1) (0.5× 106 cell into dye working solution of 500ul)
1.3 Incubate for 5 minutes at 37°C.
1.4 Centrifuge the labeled suspension tubes at 1000rpm for 4 minutes.
1.5 Remove the supernatant and gently resuspend the cells in pre-warmed (37°C) growth
medium.
1.6 Wash two more times
2. Cell fixation and nuclear staining
2.1 Dilute the MSC Attachment Solution 1:100 in PBS, coat cover slips with diluted MSC
Attachment Solution for 10 mins;
2.2 remove the MSC Attachment Solution and Seed the stained cells on the coverslips for 10mins
2.3 Aspirate the medium, fix cells with fresh-prepared chilled 4% paraformaldehyde in PBS+ of
500ul per well, approximately 10 minutes incubation time on ice.
2.4 Wash the cells with chilled PBS+ 3 times (5 min each).
2.5 Prepare working solutions: add NucBlue® Live cell stain 3drops per mL of PBS.
2.6 Remove PBS+, add NucBlue® Live cell stain working solution 300ul per well (24 well plate),
incubate 20 min at 20-25oC.
2.7 Aspirate NucBlue® Live cell stain working solution. Rinse the cells three times in PBS+, remove
PBS+, add 300ul DIH2O.
2.8 Mount the coverslips as described in Mounting Live Cells onto Microscope Slides.