The STEMdiff™ Trilineage Differentiation Kit: Assessing Differentiation to Three Germ Lineages

To assess differentiation to three germ lineages after STEMdiff™ Trilineage Differentiation, cells should be harvested and/or fixed for analysis of lineage-specific markers on day 5 for mesoderm and endoderm lineages and day 7 for the ectoderm lineage. The analysis method is determined by the user and may include flow cytometry, immunocytochemistry, or transcriptome analysis.  If assessing for differentiation by flow cytometry or immunocytochemistry, label with combinations of fluorochrome conjugated antibodies specific for early differentiation for each germ layer, for example:

  • Ectoderm: PAX6 combined with Nestin (e.g. Anti-Human Nestin Antibody, Clone 10C2, Catalog #60091)
  • Mesoderm: Brachyury (T) combined with either NCAM or CXCR4 (e.g. Anti-Human CD56 [NCAM] Antibody, Clone HCD56, Catalog #60021; or Anti-Human CD184 [CXCR4] Antibody, Clone 12G5, Catalog #60089)
  • Endoderm: SOX17 combined with either CXCR4, clone 12G5, Catalog #60089 or FOXA2

Recovering single cells for flow cytometry analysis:

The following are instructions for harvesting cells from 1 well of a 24-well plate. If using other cultureware, adjust volumes accordingly.

  1. Warm ACCUTASE™ to room temperature (15 - 25°C).
  2. Aliquot 1 - 2 mL of FACS Buffer (PBS + 2% FBS) into tubes that will be used for collecting cells.
  3. Aspirate medium from well. Wash well once with 1 mL D-PBS without Ca++ and Mg++. Discard the wash.
  4. Add 0.5 mL ACCUTASE™ per well. Incubate at 37°C for 8 - 10 minutes.
  5. Harvest cells using a 1 mL micropipette, pipetting up and down over the growth surface to break up cellular aggregates and obtain single cells.
  6. Transfer the single-cell suspension into tubes containing FACS Buffer. Rinse wells with an additional 0.5 mL of FACS Buffer and add the rinse to the tube containing the cells.
  7. Centrifuge at 300 x g for 5 minutes.
  8. Pour off and discard the supernatant. Resuspend cells in FACS Buffer.
  9. Count viable cells using Trypan Blue and a hemocytometer. The single-cell suspension may now be used for flow cytometry analysis of choice.

Flow cytometry labeling protocols:

  1. Warm sufficient volumes of 4% paraformaldehyde (PFA) Solution, Saponin Buffer (1 mg/mL saponin in PBS + 1% BSA), and FACS Buffer (PBS + 2% FBS) to room temperature (15 - 25°C).
  2. Centrifuge cells at 300 x g for 5 minutes. Pour off and discard the supernatant.
  3. Resuspend cells in ~1 mL 4% PFA Solution. Incubate at room temperature (15 - 25°C) for 15 minutes.
  4. Add 2 - 3 mL FACS Buffer to the tubes containing cells. Centrifuge at 300 x g for 5 minutes. Pour off and discard the supernatant.
  5. Resuspend cells in FACS Buffer at the desired concentration; the fixed cells are now ready for labeling.
  6. NOTE: Fixed cells can be kept at 2 - 8°C for up to 4 days prior to labeling.
  7. Aliquot approximately 3 x 10^5 cells per sample into a 5 mL FACS tube.
    NOTE: Samples requiring intracellular antigen labeling only (e.g. Ectoderm) can be set aside on ice or at 2 - 8°C. Proceed to step 12 for labeling these samples.
  8. For samples requiring cell surface antigen labeling, centrifuge each sample of fixed cells at 300 x g for 5 minutes.
  9. During centrifugation, prepare antibody solutions for labeling of cell surface antigens (CXCR4 or NCAM) and corresponding controls in FACS Buffer with a volume of 100 µL per sample.
  10. Pour off and discard the supernatant. Resuspend samples in 100 µL of the appropriate antibody solution (prepared in step 9).
  11. Incubate samples in the dark at room temperature (15 - 25°C) for 30 - 60 minutes.
  12. Add 1 mL FACS Buffer to each labeled sample.
  13. Centrifuge all samples (labeled samples as well as samples set aside in step 6) at 300 x g for 5 minutes. Pour off and discard the supernatant.
  14. Add 1 mL of Saponin Buffer to each sample. Incubate at room temperature (15 - 25°C) for 15 minutes to permeabilize cells.
  15. Centrifuge all samples at 300 x g for 5 minutes.
  16. During centrifugation, prepare antibody solutions for labeling of intracellular antigens (PAX6 + Nestin, T, SOX17) and corresponding controls in Saponin Buffer with a volume of 100 µL per sample.
  17. Pour off and discard the supernatant. Resuspend samples in 100 µL of the appropriate antibody solution (prepared in step 16).
  18. Incubate samples in the dark at room temperature (15 - 25°C) for 30 - 60 minutes.
  19. Add 1 mL Saponin Buffer to each labeled sample.
  20. Centrifuge all samples at 300 x g for 5 minutes. Pour off and discard the supernatant.
  21. Resuspend samples in FACS Buffer for analysis on the flow cytometer.

For further questions, you can please contact Technical Support at techsupport@stemcell.com.