General Extracellular Target Flow Protocol

1.    Grow cells to 50-75% confluency. Flow cytometry requires between 2 x 105 and 1 x 106 cells for optimal performance.
2.    If cells are adherent, harvest gently by washing with 1X PBS + 1% BSA (Flow Buffer) and scraping. Avoid using trypsin as this can disrupt certain epitopes of interest. If enzymatic harvest is required, use Accutase or Collagenase for a less damaging option.
3.    Reserve 100 uL for counting, then transfer cell volume into a 50mL conical tube and centrifuge for 8 minutes at 400 RCF (~1200 RPM)
a.    Count cells using a hemocytometer and a 1:1 trypan blue staining to determine the health of the cells before starting flow protocol. If cells are unhealthy per the trypan blue staining, do not proceed.
4.    Re-suspend cells to a concentration of 1 x 106 cells/mL in Flow Buffer (1X PBS + 1% BSA)
5.    Aliquot out 1 mL samples in accordance with your experimental samples.
6.    Stain each sample at 1 uL/ 1 x 106 cells of primary antibody or 1-3 uL/ 1 x 106 cells for directly conjugated antibodies. Mix well and incubate on ice for 30 min- 1 hour. Gently mix samples every 10-15 minutes
7.    Following the primary/conjugate incubation, add 2 mL/sample of Flow Buffer and centrifuge for 5 min at 400 RCF (~1200RPM)
8.    Remove supernatant and re-suspend each sample in 2 mL Flow Buffer, repeat wash for 5 minutes at 400 RCF (~1200 RPM)
9.    The next step differs based on experimental design:
a.    If using a fluorescent secondary, re-suspend the pellet in 1 mL Flow Buffer and add 1 uL secondary to each sample. Mix and incubate on ice for 20 minutes.
b.    If using a directly conjugated antibody, after the 2nd wash, re-suspend cell pellet to a final volume of 500 uL per sample and proceed with flow analysis.
10.    In the primary-secondary experimental design, following the 20 minute secondary incubation on ice, add 1 mL Flow Buffer to each sample and centrifuge for 5 minutes at 400 RCF (~1200 RPM)
11.    Remove supernatant and add 2 mL Flow Buffer per sample and repeat wash for 5 minutes at 400 RCF (~1200 RPM)
12.    Re-suspend cell pellet to a final volume of 500 uL in Flow Buffer. Proceed with flow analysis.