Samys Stromal Cell Staining Protocol
Disclaimer: This protocol is quite thorough, yet you may be required to perform additional steps that are not listed below. In any case where there is doubt ALWAYS ask someone, and if there is no one to ask try and use common sense.
- Check/order mice at specified age, well in advance, and check again day before experiment.
- Book FACScalibur and microscope/coulter counter in advance.
- Make up antibodies and enzymes coll/disp and Dnase the previous day at CORRECT dilutions.
- Label V bottom tubes/FACS tubes, yellow capped tubes, etc.
- Cut up pieces of mesh for filtering
- Prepare several different bore sized pasteur pipettes.
- Type out/write protocol and show to stromal experts.
- Before removing thymii clean dissecting equipment, set up petri dishes and place media in it.
- Remove thymii from mice and remove excess fat/connective tissue.
- Make three to five 2-3mm incisions in thymii before putting in bottle with 50ml media with flea.
- Place on magnetic stirrer incubating at 4 degrees for 40 mins (or 2 x 20min incubations). If you are using many thymii, you may need a longer incubation period for thymocyte depletion.
- Place 30ml of RPMI medium into a 50ml tube (label collagenase/dispase) and 5ml of RPMI medium into a 10ml tube (label Dnase)
- Make up enzymes into plastic weighboats.
- Turn on water bath.
- Get ice bucket.
- Enzymes are to be made at the following concentrations:
0.125% collagenase/dispase = 0.0125gx3 = 0.0375g into 10ml x 3 = 30ml RPMI
0.1% Dnase = 0.005g into 5ml RPMI
Thymic Enzymatic Digestion
- After 40 min incubation is over, transfer 30-35ml of media from bottle into a 50ml tube, ensuring thymii are not poured in.
- Pour remaining 15ml into a petri dish, get rid of excess media. Break a glass pipette so that it has a wide bore so you can pick up thymii and put into a 10ml tube.
- Agitate thymii carefully with broken pasteur pipette several times before removing supernatant. Then add 4.5ml collagenase/dispase and 0.5ml Dnase to yellow capped tube.
Thymic Agitation Digestion
- Place tube in 37 ° C water bath for 15 minutes, resuspending every 5 minutes.
- Slowly decrease the bore size of the pasteur pipette after or within each digest.
- Place supernatant into another yelow capped tube.
- This constitutes digest 1.
- Resuspend thymii with 4.5ml Collagenase and 0.5ml Dnase and place in water bath again.
- Decrease the bore size of the pasteur pipette unti fragment are small enough to be resuspended through a full size pasteur pipette.
- One should resuspend SLOWLY, VERY SLOWLY using an 18G needle, and slowly progress to a 21G followed by a 23G needle.
- Avoid using a 26G needle at all costs.
- Repeat until thymii have been digested, usually 4-6 digests required.
- Place a few drops of digest 1 and look at the thymocytes:stromal cell ratio.
- The first digest will usually consist of thymocytes only.
- Place digest on ice.
- Spin digest straight away, remove supernatant (to get rid of enzymes) and resuspend in 5ml PBS/FCS/azide (to stop cell cycle).
- Leave on ice.
- Repeat Thymic Agitation Digestionsteps until thymii are digested.
- Looking at thymocyte to stromal cell ratio throughout and between each digest.
- Whilst stromal cells can be the same size as lymphocytes, they are generally larger and are therefore easily distinguishable under the microscope.
- Perform cell counts on digests 4-6 and determine total cell number and desired number of cells.
- ie: 38 tubes with 3 x 10^6 cells/tube = 114 x 10^6 = 1.14 x 10^8 cells, therefore we need at least this number of cells.
- One may have enough cells in digest 5-6 or 4-5 (depending on which is stromal cell rich) and therefore only need to pool these digests.
- Pool 3 digests (in 10ml tubes) into one 50ml tube and run through 100 micron strainer/mesh to remove any large particles
- centrifuge and resuspend in desired volume.
Eg: 38 tubes total:
We want 38 tubes with 100 m l in each.
Therefore we need 38 x 100 = 3800ul = 3.8ml TOTAL
We have _____________ total number of cells
_______ / 3.8 ml = __________cells/ml
_________ cells in 1000ul
_________ cells in 100ul
Therefore in each 100 microlitres I will have _________ cells
Thus one can spin cells down and resuspend in 3.8mls to get 38 tubes of 100ul which has ____________cells , or to get a more dilute amount resuspend in a greater volume.
- Distribute 3x10^6 cells into each V bottom FACS tube, filtering through a mesh.
- Spin for 5 minutes.
- Resuspend in FACS buffer/RPMI or antibody.
Things To Consider
- Once cells are spun, ensure that they are not sitting in pellet for too long (no longer than 5 minutes or else cell viability will be affected-big time).
- Apart from when you are digesting thymii or you explicitly need to, all manipulations should be performed at 4 ° C.
- If you are using embryonic (E13-E21) thymii, fewer digestions will be required since there are fewer cells,
- Often 1-2 digestions will suffice.
- Be very careful with thymii of this age, as they are very sensitive and can tear easily as there is little fat/connective tissue on them
- Hence mechanical digestions should be performed with a great deal of care.