Metastatic Matrigel Invasion Assay (Dr. Kirshner Lab)-Teelab
A.Barnard
Noted Materials
5ml and 10ml Pipettes in 4C
10ul, 200ul, and 1000ul pipette tips in 4C
Matrigel in -80C, thaws overnight at 4C
24-well cell culture plate and 8.0micron transwell inserts
Protocol/Solutions
Polymerized Collagen 1 Gel (2mg/mL)
-Make this solution fresh EACH experiment
-Calculate total volume needed for full experiment and use 20-30% extra since very viscous,
assure slow pipetting to get all of solution
-Neutralization buffer:
-Add 100mM HEPEs to 2x PBS on ice
Assure pH is between 7.0-7.4 (rarely needs adjustment)
-Dilute concentration of rat tail collagen 1 in neutralization buffer to a 2mg/mL concentration
and vortex at low speed
-Stock lasts for 6months in -20C
-ADD 2mg/mL collagen 1 gel solution to bottom of plate well, spread around
-Incubate in 37C for 1+ hour(s) for polymerization
Reconstructed Endosteum (rEnd) (5mL)
Components
Stock
volume taken Final conc.
Ratio of
concentration from stock
(approx..)
components
1 mg/ml
384.6 µl
77 µg/ml
5.32 parts of FN: 1
Fibronectin
part of collagen I:
72.3 µl
29 µg/ml
Rat tail Collagen type 2 mg/ml
62.8 parts of PBS
I (solution no. 1)
1X
4.543 ml
1X
Sterile PBS
-Fibronectin from powder, good for 6-12 months in 4c, or freeze in -20C
-Polymerized collagen type I is from the prior solution
-AFTER polymerized collagen gel1 has completed its incubation, add 130ul of the rEnd to each
well (24-well plate)
-Spread with pipette tip “color well” but do not puncture lower gel layer
-Incubate in 37C for 1hr
Split and count cells with Tryphan Blue
-Seeding density for 24-well plate, calculate to 25,000 cells/transwell
-Overestimate cell counts to assure enough
-Resuspend cells in PBS, NOT media for final count
-Pipette final volume (25,000 cells/7ul PBS) of needed cells into master 1.5mL tube on ice
Reconstructed Bone Marrow Matrix w/o Hyaluronic Acid (rBM) (5mL)
Components
Stock
volume taken Final conc.
Ratio of
concentration from stock
(approx..)
components
5 ml
3.72 parts of
Matrigel
matrigel: 2.43 parts
1 mg/ml
3.27 ml
340 µg/ml
Fibronectin
of fibronectin: 1
2 mg/ml
1.345 ml
280 µg/ml
Rat tail collagen I
part of collagen I
-Add matrigel first, using 1000ul tips (lower surface area = lower dilution), never reuse tips
-Assure no bubbles in matrigel!
-Matrigel will change to peach color upon addition, assures proper pH
-Store in 4C if not immediately using rBM solution
-Keep any extra in 4C for 2-3 days, otherwise make fresh each time
-AFTER rEnd incubation is complete, remove extra rEnd solution from each well via aspiration
-ADD 75ul rBM to the center of each well quickly and evenly layered across full plate
-Spread matrix with pipette tip, “color well” lightly (light pink color)
-Incubate in 37C for 1hour
Transwell Set Up
-Use another empty 24-well plate for transwell set up
-Place one transwell per well using autoclaved sterile foceps, only touching tabs on outside top
-Each sample has own 1.5mL tube on ice (pre-cooled)
-Add 23ul matrigel into each tube (never reuse tips!)
-Add 7ul of the cell-PBS mixture to each tube
-Mix matrigel-cell-PBS mixture up-down ONE time with pipette tip, NO BUBBLES
-Add mix to center of transwell, carefully spread, no bubbles in middle, quickly add
-Incubate transwells in 37C for 30+minutes
Bone Marrow Growth Media (BMGM) (500mL)
Stock
Working
Vol for 500ml
Component
concentration concentration medium
6.2 x 10-4 M
CaCl2
0.62 M
500 µl
(1:1000)
Sodium
10-6 M
10-3 M
500 µl
Succinate+Hydrocortisone
(1:1000)
Penicillin-Streptomycin
1%
5 ml
FBS
20%
100 ml
RPMI 1640
394 ml
-Warm in waterbath to 37C (cooler media could disturb matrigel)
-BMCM: 80% conditioned media (BMGM in bone cells for 3 days, removed, spun, filtered, frozen)
+ 20% fresh BMGM
-BMCM from Dr. Kirshner lab only
-Must also be warmed to 37C
-Stored in 4C
-AFTER both incubations complete, add set transwells into plates using sterile forceps
-ADD 1mL warmed BMCM to BOTTOM of each well by dripping slowly down side of well
Mammary Epithelial Growth Medium (MEGM)
Stock
Working
Volume for 500ml
Component
concentration
concentration medium
RPMI 1640
490 ml
Horse serum
1%
5 ml
Penicillin streptomycin
1%
5 ml
-Warm in waterbath to 37C
-Stored in 4C
-Add 0.5mL to TOP (aka transwell) of each transwell by dripping slowly down side of transwell
Culture Maintenance
-Day 7: add 0.2mL fresh warm MEGM to transwell top OR do 50:50 media change for both
MEGM and BMCM
-Remove transwells and place in empty plate to change bottom media (only
touch sides with sterile forceps)
-Do not touch matrix or membrane!
Imaging
-See fluorescent imaging protocol for more details
-Culture cells for a total of 14 days from day of plating
-Image every few days with fluorescent scope (if using GFP cells).. do not mistake pores for cells!
-Remove transwells to image bottom cells
-Re-add transwells to image top cells
-Microscope use (Hansen, Lab#235):
-Day 14: take final images and move to processing and analysis