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HOME > Protocols > Cell Biology > Cell Culture Protocols > Protocol for Preparation of Splenocytes

Protocol for Preparation of Splenocytes

  1. Place the spleen in 3ml of media (i.e. RPMI 1640 with 10% FBS) in a small petri dish (35x10mm)
  2. Using sterile forceps, place the spleen on a sterile wire mesh screen (200um bar width and 340um open space)
  3. Push the spleen through the screen with the plunger of a 10ml syringe into the petri dish from step 1. Rinse screen with the 3ml of media. The capsule of the spleen will be retained by the screen. It is important to minimize the amount of capsule in the media as this interferes with subsequent purification, so don't push too hard!
  4. Bring the spleen mixture up to 8ml with the same type of media used in step 1
  5. Mix the suspension by gently pipetting up and down
  6. Carefully layer the solution over 3ml of Lympholyte-M (specific for mouse). Do not allow the layers to mix since the cells will be retained at the interface of the two layers
  7. Centrifuge at 500rcf for 30 minutes
  8. Remove cells from the white buffer coat layer which will probably appear to be a slightly diffuse layer of cells at the interface of the Lymphocyte-M and media
  9. Bring the cells to 8ml with the same type of media used in step 1 and centrifuge 10 minutes at ~180rcf to wash and pellet the cells
  10. Discard supernatant
  11. Resuspend the cell pellet in 10ml of the same type of media used in step 1
  12. Make 1ml of a 1:10 dilution of the suspension for step 10 and count cells using a hemocytometer or cell counter
  13. Centrifuge cells from step 10 for 10 minutes at ~180rcf
  14. Discard supernatant
  15. Resuspend cells at working concentration (typically 2 x 106 / 100ul)

Note: To properly seed a 96 well plate for growth, place approximately 2.5 x 105 cells per well.

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