In vivo cell monitoring - BD Pathway Bioimager 855 (JUMC)

From LipidomicsWiki

Visualization and in vivo monitoring of live cells by BD Pathway Bioimager 855

Cell types: HUVECs, skin fibroblasts

1. Seed cells in 96-well clear bottom tissue culture plates for imaging applications (Becton Dickinson; Cat. No. 353119) at 10000 cells per well (starting recommendation)
2. Culture cells overnight (16 hours) in standard conditions to achieve 50% confluency (the initial number of cells per well and incubation time should be optimized empirically to account for specific growth rate differences between cell types so that the cell density optimal for image acquisition is not compromised)
3. Remove medium, wash cells with 100 µl PBS, replace fresh medium
4. Add: Hoechst 33342 (Molecular Probes, Cat. No. H-3570) – final concentration of 2 ng/µl and TMRM (molecular Probes, Cat. No T-668) – final concentration of 20 µM
   
5. Incubate for 1 hr at 37oC
6. Remove medium, wash cells with 100 µl PBS, replace fresh medium
7. Image cells using the 20x/NA 0,75 objective and AttoVision software (select Hoechst for nuclear ROIs, and TMRM for cytoplasmic ROIs; avoid prolonged exposure to light to prevent bleaching)
8. Repeat imaging serially as required; the number of scans and intervals between scans should be adjusted individually for each cell type and experiment setting; as a general rule, balance the predicted time period of the monitored process (e.g. loss of mitochondrial membrane potential) vs the photobleaching effect (too frequent scans may substantially reduce the signal before the monitoring is complete).