Persomics Lab Protocol Template

The following protocol is followed when screening at Persomics labs. Note that this protocol should be adapted to the conditions of your assay and specific cell lines.

Disclaimer: The publication of this protocol template does not constitute a guarantee on the part of Persomics and its affiliates shall not be liable for the results in individual experiments.

Always adhere to the safety protocols prescribed by your lab.

Recipes for Antibody and Staining Solutions

Primary Antibody: Rabbit IgG 1:2000 in 10% goat serum diluted with PBS 

Secondary Antibody: Fluorescent Anti-Rabbit IgG 1:1000 in 10% goat serum diluted with PBS 

Hoechst: 10mM Hoechst 33342 in DMSO diluted 1:1000 in PBS

Fix: [4.0% w/v Paraformaldehyde in PBS pH 7.4]

Perm: [0.1% v/v TritonX-100 in PBS]

Blocking Buffer/Goat serum: [10% v/v goat serum in PBS]

 

1. Tissue Culture

Follow basic tissue culture protocol:

  1. aspirate media,
  2. rinse with PBS,
  3. trypsinize, incubate, and detach cells,
  4. add media,
  5. split to new flasks.

Note

Use appropriate trypsin, media, and incubation parameters for specific cell line.

2. Plate cells

  1. observe cells for viability and ~95% confluency
  2. aspirate media
  3. rinse with PBS
  4. add trypsin and incubate
  5. check for detachment
  6. add OptiMEM
  7. perform cell count using both chambers of hemacytometer
  8. determine the volume required to obtain ~1.5 million cells
  9. plate cells to 15 mL total per array with OptiMEM
  10. swirl in a figure eight to coat
  11. incubate 48-96 hours (37°C / 5% CO2)

3. Stain & image

  1. observe cells on arrays for viability and confluency
  2. aspirate media
  3. rinse with 10 mL PBS
  4. add 10 mL Fix and put on shaker (15 rpm) for 10 min
  5. aspirate Fix and rinse with 10 mL PBS
  6. add 10 mL Perm and put on shaker for 10 min
  7. aspirate Perm
  8. add 10 mL primary antibody in goat serum and put on shaker for 60 min
  9. rinse 5x with 10 mL PBS on shaker for 5 min
  10. add 10 mL secondary antibody in goat serum and put on shaker for 30 min
  11. rinse 5x with 10 mL PBS on shaker for 5 min (each rinse)
  12. add 10 mL Hoechst and put on shaker for 10 min
  13. rinse 2x with 10 mL PBS
  14. add 10 mL PBS
  15. image arrays (*10-20X)

Note

Use appropriate PBS for specific cell line. Aspirate and pipette on corner of plate. DO NOT TOUCH GLASS!