Cell Surface Stain Protocol

Cell Surface Staining

The following protocol is intended for PBMC or spleen cells but can be adopted for any types of cell. Harvest cells in FACS stain buffer (BD, 554657) alternately you can use DPBS with 0.2% BSA as stain buffer. Count the cells.

  1. Take cells between 1 to 10 million in a FACS tube in less than 100 µl volume
  2. Add 5 µl of FC block (BD, 564219 or 553141 for Human or mouse)
  3. Add 5 µl of True-Stain Monocyte Blocker (Biolegend, 426102), only if you wish to analyze Monocytes
  4. Incubate for 10 min at RT
  5. Add the anti-body cocktail (only in special cases a specific Ab need to be added separately)
  6. Add stain buffer up to 200 µl
  7. Incubate at RT for 20 minutes in dark
  8. Add 3 ml of cold PBS/DPBS, quick vortex
  9. Spin for 5 minutes, 400g in cold
  10. Decant supernatant
  11. Wash again
  12. Resuspend in 200 µl of DPBS containing Live-Dead dye
  13. Incubate at RT for 15 minutes in dark
  14. Add 3 ml of cold stain buffer, quick vortex
  15. Spin for 5 minutes, 400g in cold
  16. Wash again using stain buffer
  17. Resuspend in 300 µl of stain buffer

How to prepare Ab cocktail

If you are using more than one Brilliant dye or Super Bright dye, you have to start with the brilliant buffer (BD, 563794). If this is not the case, then this buffer can be ignored. Below recipe is formulated for one staining.

  1. Add Brilliant stain buffer (10 µl for 100 µl of final stain volume)
  2. Add all the Abs as decided by individual titration
  3. This mix can stay fully functional for few weeks, store at 4oC, dark, make sure do not freeze

For multiple use just increase the volume of all the components accordingly.