Tissue Dissociation Protocol Reference Guide
Note: Please contact email@example.com if you are looking for a dissociation protocol for mouse CD11b microglia.
Note: Enzymatic digestion is only recommended to isolate CD11c cells downstream using EasySep isolation kits.
Note: This protocol is recommended for most mouse EasySep isolation. For more details please see complete protocol "Mouse Spleen Dissociation Protocol" below
Note: For more details please see Mouse Spleen Digestion Protocol as noted below
Mouse Spleen Mechanical Digestion ProtocolThis protocol will outline how to harvest cells from a spleen sample, and prepare a single cell suspension prior to performing cell isolation. Preparing a true single cell suspension of the primary tissue sample will optimize cell separation by avoiding additional cell loss and enabling maximum labeling of the target cells.
- Transfer the spleen to be processed into a sterile 35 mm culture dish (Cat #27100/27150) containing 5 mL of recommended medium for downstream isolation, this will be indicated on your kit PIS. If you are not using an isolation kit following dissociation please use PBS + 1 mM EDTA.
- Note: If processing multiple spleens please use a 100 mm culture dish (Cat #27110) containing 10 mL of recommended medium.
- Trim any extra connective tissues or fat from the spleen, take note of any necrotic regions, and inspect the spleen for any other phenomena such as enlargement, discolouration, or lesions. It is important to note the condition of your starting sample as this may be taken into consideration when evaluating your cellular fraction.
- Take the piston/plunger out of a sterile 3 cc syringe (Cat #28230/28240). Use the flat back end of this device to mince the spleen by crushing the spleen 5 times in gentle circular motions. This action will burst the spleen, disrupt the pulp, and release the splenocytes.
- Over a sterile 50 mL conical tube, place a 70 μm cell strainer (Cat #27216/27260) and pass 2 mL of recommended medium through to prime the filter.
- Note: Priming the filter reduces cell adhesion that may occur if the filter is dry when cells are passed through.
- Triturate the released splenocytes to a homogeneous mixture with a primed 5 or 10 mL serological pipette.
- Transfer the supernatant and tissue from the culture dish to the primed 70 μm cell strainer (Cat #27216/27260). Using a fresh piston/plunger out of a sterile 3 cc syringe gently pass the cells and the dissociated tissue through the cell strainer membrane by pressing in a circular motion with the plunger/piston end of the syringe. Wash the strainer with 3 mL of recommended medium.
- Optional high recovery protocol:
- Do not transfer the spleen tissue to the strainer. Instead add an additional 5 mL of recommended medium to the dish containing the spleen.
- Note: If processing multiple spleens use 10 mL of recommended medium
- Repeat steps 4-6 and remember to add back recommended medium (5 or 10 mL) until the spleen tissue appears white and buffer runs clear
If you have any further questions about these tissue dissociation protocols, please feel free to contact Product and Scientific Support at firstname.lastname@example.org.