adapted from Pullan, S.E. and Streuli, C.H. (1996). The mammary gland epithelial cell. In Epithelial cell culture Harris, A. (ed., pp 97-121. Cambridge University Press, Cambridge, UK
| Dissecting tools | Autoclaved |
| cotton swabs | Autoclaved |
| Sterile scalpels/razor blades | disposable or Autoclaved |
| F12 media* | (Invitrogen) 4°C |
| F10 media* | (powder, Invitrogen) 4°C |
| Fetal Bovine Serum (FBS) | JRH biosciences -20°C |
| Insulin stock | 1 mg/ml in 0.01N HCl -20°C |
| Hydrocortisone stock | 1 mg/ml in ethanol -20°C |
| EGF stock | 10μg/ml in H2O -20°C |
| gentamycin | 10 mg/ml (Sigma or Invitrogen) 4°C |
| Pen/strep | 10,000U (Invitrogen) -20°C |
| Fetuin | Sigma F3385 4°C |
| Collagenase A | Roche 1088793 (1088795) 4°C |
| Trypsin | Invitrogen 27250-042 4°C |
| Tissue culture plates# | 100mm~80cm² |
| 6 well ~ 10cm² | |
| 12 well ~ 0.5cm² | |
| poly-lysine coated coverslips | |
| miscellaneous disposables | (50 ml tubes, eppendorf tubes, pipettes, pipette tips, etc.) |
–We have used DMEM/F12 (Invitrogen) instead of F12 and/or F10 as well
— I usually plate on 6 well plates and use one well to count cells to determine cell number for infection with Adenovirus. Several wells are pooled for transplantation or other procedures.
37°C shaking incubator
Flow hood
Tissue culture incubator 37°C/5%CO2
Tissue culture hood
- Autoclave dissection tools, cotton swabs.
- Reserve hood and incubators.
- Reserve 37°C incubator/110-125RPM.
- Prepare wash buffer:
500ml F12
2.5ml gentamycin (50μg/ml)
25ml FCS (5%) - Serum/Fetuin to coat plates at 100μl/cm².
20% FCS
2mg/ml Fetuin in F12
Filter sterile and store at 4°C. - 2x hormone plating media:
Insulin 10 μg/ml (stock 1mg/ml) (1ml/100ml)
Hydrocortisone 2 μg/ml (stock 1mg/ml) (200ul/100ml)
EGF 10ng/ml (stock 10μg/ml) (100ul/100ml)
Gent. 100 μg/ml (stock 10mg/ml) (1ml/100ml)
Pen/Strep 200 U/ml (stock 10,000u/ml)(2ml)
Bring up to volume in F12 media
Filter Sterile, store at 4°C.
No FCS! (Already in serum/fetuin mix) - Growth media
Insulin 5 μg/ml (stock 1mg/ml) (500ul/100ml)
Hydrocortisone 1 μg/ml (stock 1mg/ml) (100ul/100ml)
EGF 5 ng/ml (stock 10μg/ml) (50ul/100ml)
Gent. 50 μg/ml (stock 10mg/ml) (500ul/100ml)
Pen/Strep 100 U/ml (stock 10,000u/ml) (1ml/100ml)
FBS 5%
Bring up to volume in F12 media
Filter Sterilize, store at 4°C.
- Make collagenase solution (Digestion medium)
Need to use 10ml/gm tissue; You get roughly about 0.5gm-2gm per mouse based on the age of mice and the number of glands taken out.
DMEM:F12
100 u/ml pen strep (1ml)
100 μg/ml gentamicin (1ml)
2mg/ml collagenase (200mg) From Roche Cat#11088793001
100u/ml hyaluronidase (7.3mg) From Sigma Cat# H3506 - Add serum Fetuin to plates at 100 μl/cm2
~8ml/ 100mm plate
~1ml/ well of 6 well plate
Incubate 4-5 hours at 37°C before plating. - Weigh 50ml tubes + 30ml wash buffer and place on ice.
- Start dissection: Place dissected tissues in 50ml tubes containing wash buffer at 4°C
- Weigh tubes containing wash buffer + harvested tissue. Subtract original weight to determine the weight of the mammary glands.
- Using sterile technique, mince tissue into very small pieces (<1mm) with a scalpel or razor blade. Place minced tissue in digestion medium (1gm tissue / 10ml medium) and shake at 37°C for approximately 2-3 hrs. The shaker should be set at 110 to 125rpm.
Small volumes can be dissociated in a 50ml tube placed at a 45° angle. (give it a good, hard shake every hour) - Follow spin scheme from here on; All subsequent spins are done in 35ml total volume, brought up to volume with wash buffer.
Sup: Spin sup at 800 rpm 3min in 35ml Pellet #1
Sup: spin 1500 rpm for 10min Pellet #2
(Discard Sup)
Combine pellet #1 and 2 in a clean tube and wash 5 times at 800 rpm for 3min in 35ml wash buffer, until buffer is clear. - Count and resuspend in 2.5 x 106 per ml in 2X plating medium.
- Plate cells at 2.5 x 105/cm2
- Add enough 2x plating medium to the culture plates to make a 1X; ie. If you had added 8ml of serum/fetuin to the plates, then add 8ml 2x hormone plating medium (including the medium added to cells).
- Incubate plates at 37°C for 2 days (36-48 hours) before further manipulations.
- After 2 days (36-48 hours) change media to growth media.
• Two to three days after the primary culture, trypsinize the cells using 0.25%
Trypsin for about 5minutes. Wash the cells in DMEM+ medium once and
then count the cells. Bring up the cells to make a 1×106cell per ml
suspension in DMEM+. DMEM+: DMEM 2% FBS 10mM HEPES
• Add Hoechst dye (Sigma cat#B2261; make a stock at 200X) at 5μg/ml final concentration. Place the cells in 37°C and mix every 10 minutes for 60-90 minutes.
Note: I have been doing mine for 60 minutes but 90 minutes has been done in other studies and in the hematopoietic system. (It is very important that the tube with cells is gently shaken every 10 minutes and the temperature kept at 37°C the whole time, do not let anyone else use the water bath while Hoechst staining is in progress).
• After the staining is completed all the subsequent washes should be done in ice cold HBSS+
Note: I usually wash once with HBSS+ at 10x the staining volume, ie if staining buffer was 5ml; add 50ml of ice cold HBSS+ and wash once. The washes can be done at 4°C, but all the other times keep the cells on ice.
HBSS+: HBSS (with Mg+ and Ca+)
Note: Bone marrow staining is done without Ca+ and Mg+, but it has been harsh to the mammary gland cells.
2% FBS
10mM HEPES
• After the wash, bring up the cells to 107 to 108 cells per ml and add 2μg/ml of propidium iodide (We usually make a 200X PI stock solution).
Please also see Dr. Peggy Goodell’s protocols for more details on how to set up the flow cytometry analysis (http://www.bcm.edu/labs/goodell/index.cfm?PMID=17588)
