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Box 1. Preparation of bovine thymocytes

From the following article

Flow cytometry and FISH to measure the average length of telomeres (flow FISH)

Gabriela M Baerlocher, Irma Vulto, Gary de Jong and Peter M Lansdorp

Nature Protocols 1, 2365 - 2376 (2006)

doi:10.1038/nprot.2006.263

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  1. Arrange for fresh bovine thymus through a local meat packer. Request the collection of 2–3 small (e.g., cubic inch) pieces in a 1,000 ml container filled with digest solution. Arrange for pickup and process immediately.
  2. Using a Petri dish lid as a cutting plate, place several pieces of thymus from the container into the lid. Pour about 20 ml of digest solution into the other half of the Petri dish containing the strainer. Using a scalpel, remove all white meat (fat) from the thymus. Cut the thymus pieces into smaller pieces about 2–5 mm in size. Put the smaller pieces in the strainer and gently force the tissue using the syringe top through the strainer (do not press too hard). Every 15 seconds, wash the strainer and tissue with digest solution.
  3. Transfer the cell suspension from the Petri dish to 50 ml falcon tubes, using a funnel. Collect about 24 tubes. Observe cells through a microscope. Most of the cells should be single, with very little clumping. If too many clumps are present, repeat the strainer step (2).
  4. Spin for 10 min at 450 g, aspirate supernatant and gently resuspend some of the cells in 10–20 ml PBS with DNase. Do not resuspend the entire pellet, as the bottom will contain most of the clumped cells. Top up the tube with regular PBS up to 50 ml. If there is still a pellet on the bottom but you resuspended lots of cells, decant the suspension into a beaker. As clumps sink rapidly to the bottom, single cells can be enriched by collecting the supernatant after 1–2 min of sedimentation.
  5. Pool 400 ml cell solution into a 1,000 ml beaker and add 400 ml of 0.2 % formaldehyde (prepare just before use by adding 20 ml 37–40% formaldehyde to 380 ml PBS). Mix gently. Put on the shaker for 10 min. Aliquot into 50 ml falcon tubes. Spin for 5 min at 450 g at 4 °C. Aspirate the supernatant and resuspend the pellets in up to 50 ml PBS. Spin for 5 min at 450g. Aspirate the supernatant and resuspend the pellet in 10 ml PBS per tube. Pool the cells in a beaker. Remove an aliquot for cell counting.
  6. Dilute with PBS so that you have 5 times 107 cells per ml. Take the same volume of ice-cold 80%FCS/20% DMSO andmix. Aliquot into cryovials so that there is 1 ml per vial.
  7. Freeze at -70 °C or lower. Include some unfixed cells to examine the effect of fixation on the hybridization efficiency.
    PAUSE POINT Fixed and frozen bovine thymocytes can be stored for up to 2 years.