CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS
· Store LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit in freezer (-5 to -30°C), desiccate, and protect from light.
· Amine reactive dyes in solution will lose their effectiveness over a short period of time, therefore it is recommended to completely use the vial once rehydrated. If this is not possible, aliquot the vials in small volumes and store at -80°C, avoiding freeze-thaw cycles.
· Volumes listed herein pertain to 12 well Transwell Inserts. Volumes may be adjusted according to approximate growth surface area when using alternate culture dishes.
· To properly analyze results, it is necessary to include a dead cell control and an unstained control.
Preparation of LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit
1. Thaw Reagent A and add 50 µL DMSO (Component B) to make a 1 mM stock.
2. Resuspend by pipetting up/down gently and protect from light on ice before use.
3. Make 1 µM working stock LIVE/DEAD solution in PBS.
1. Generate dead cell control
a. In a 15mL Falcon tube, collect a cell pellet of 1x105 – 1x106 cells of the cell type(s) being exposed in Step 2.
b. Resuspend each cell pellet in 1mL 4% paraformaldehyde in 1X PBS.
c. Fix cells for 15 minutes at room temperature.
d. Centrifuge tubes at 2000 RPM for 4 minutes.
e. Wash pellets twice by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container.
f. Cell pellet can be kept at room temperature when using day-of or frozen in the -20°C until ready to be used.
i. NOTE: Cells frozen in the -20°C should be used within a week.
2. Aspirate media from cells of interest and cells plated for the unstained control. Rinse cells once in PBS and trypsonize cells using 300µL of trypsin and 150µL of trypsin in the basolateral and apical compartment of TranswellÒ inserts, respectively. Incubate for 4 minutes at 37°C.
a. NOTE: incubation times will need to be optimized for different lots of trypsin
3. Add 1mL and 500µL of appropriate cell media to the basolateral and apical compartment of TranswellÒ inserts, respectively, and collect cells into 15 mL Falcon tubes. Rinse wells and inserts with 1 mL of appropriate cell media and add to respective Falcon tubes.
4. Centrifuge tubes at 2000 RPM for 4 mins.
5. Aspirate media from pellets and resuspend the unstained control pellet in 1mL PBS. Resuspend the remaining cell pellets and dead cell pellet in 1 mL of 1 µM working stock LIVE/DEAD solution.
6. Transfer resuspended pellets to 5mL Round-Bottom tubes.
7. Incubate tubes for 30 mins at 37 oC protected from light.
8. Proceed to flow cytometry on BD FACS.
Flow Cytometry on BD FACSLyric™
1. Follow start-up protocol – warm up lasers, Daily Clean, and Qualitative Control. Near-IR fluorescent probe has an excitation of 633/635 nm and emission of ~780 nm for use with the APC-Cy5 filter.
2. Insert vehicle tube and select Preview to view side (SSC) and forward (FSC) scatter pattern for P1. Select APC-Cy7 vs. FSC for P2 to draw positive gate.
3. Insert Dead control and select Preview to view side (SSC) and forward (FSC) scatter pattern for P1. Select APC-Cy7 vs. FSC for P2 to draw negative gate and confirm positive gate.
4. Acquire 10,000 events in P1.