Equipment and Supplies
 Centrifuge
 Serological pipettes (2, 5, 10, 25, and 50 ml)
 Micropipette and tips (10 μ L, 1000 μ L)
 Aspirating pipets – 9 inch – Glass Pasteur (Fisher Cat#1367820D)
 15 ml conical tubes
 50 ml conical tubes
 Accumax (Sigma Cat#A7089)
 Cell Scraper (Greiner Bio-One #541 070)
 1.5 ml Microcentrifuge tubes
 Falcon Cell Strainer - 40 μ m nylon mesh (VWR Cat#21008-949)
 Tissue culture treated sterile plates
 Matrigel® – Growth Factor Reduced – 10 ml (Corning Cat#356231)
 DMEM/F12 (Life Technology Cat#11330032)

RPE media [DMEM/F12 (70%/30% v/v) with 1% FBS and 1% Anti/Anti]
We recommend making RPE media from:
 DMEM 1X – 500 ml (Life Technologies Cat#11960044)
 Ham’s F12 – 1000 ml (Life Technologies Cat#11765047)
 Antibiotic/Antimycotic (Anti/Anti) – 100X (Life Technologies Cat#15240062)
 Fetal Bovine Serum (FBS) – Heat Inactivated – Cell culture grade (Sigma
Cat#F4135)
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1. Thaw 10 ml stock vial of Matrigel® at 4°C overnight. Put 1000 μ L pipette tips and sterile 1.5 ml microcentrifuge tubes in the refrigerator at the same time to cool down to 4°C.
2. Remove Matrigel® from the refrigerator and place on ice along with 10 x 1.5 ml microcentrifuge tubes.
3. Place your ice bucket in a tissue culture hood and pipette 1 ml of Matrigel® into each of 1.5 ml microcentrifuge tube.
4. Close the microcentrifuge tubes and immediately place in a -20°C freezer until ready for use.
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1) Aspirate off old RPE media.
2) Cover cells with 2.5 ml of Accumax.
3) Incubate at 37°C for 30 minutes.
4) Remove from incubator and add 1.5 ml RPE media.
5)
Gently
scrape all cells off the flask with a sterile cell scraper.
6) Place cell suspension in a 15ml conical tube.
7) Rinse the flask with 6ml RPE media and add to the 15ml tube containing the cell suspension.
8) Centrifuge cells for 5 minutes at ~450 x g.
9) Discard supernatant.
10) Resuspend the cell pellet in 2.0 ml of Accumax and pipette up and down using a
2.0 or 5.0ml serological pipette, gently , to mechanically break up any large clumps of cells.
11) Pipette cell suspension into an uncoated flask or plate (NO Matrigel®).
12) Incubate for 20 minutes at 37°C.
13) Remove flask/plate from incubator and gently pipette up and down with a 1000 μ L manual pipette to break-up any large cell clumps
14) Examine the cells under the microscope to ensure they are mostly single cells. If there are still large clumps return the plate to the incubator for another 10 minutes.
15) Dilute the cell suspension with RPE media to ~10ml and transfer to a 15 ml conical tube. Centrifuge for 5 minutes at ~450 x g.
16) Discard supernatant
17) Resuspend cell pellet with 4 ml RPE media
18) Filter the 4 ml cell suspension through a 40 μ m nylon mesh filter into a 50 ml conical tube. To collect remaining cells, rinse the 15 ml conical with 3ml of RPE media and put through nylon filter.
19) Once the mesh cell strainer is empty discard it and rinse the sides of the 50ml conical with 3ml media.
20) Transfer the cell suspension to a fresh 15ml conical for centrifugation.
21) Centrifuge at 450 x g for 5 minutes.
22) Discard supernatant.
23) Suspend cells in 1 ml RPE media.
24) Count cells with a hemocytometer or other cell counting device.
25) Coat plates, permeable supports, coverslips, etc. with Matrigel® as described below.
26) Plate cells on at 400,000 cells/cm².
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This protocol is for standard tissue culture plates and flasks.
*Note: Matrigel® is temperature sensitive and forms a gel when warmed to room temperature. To achieve optimal coating, work quickly, keep plates and reagents on ice, and use chilled pipettes and pipette tips to keep the Matrigel® cool.
1. Thaw a 1ml aliquot of Matrigel® at 4°C overnight. Put all plates and pipettes in refrigerator at the same time to cool to 4°C.
2. Place 4ml ice cold DMEM/F12 in a 50ml conical tube and place the tube on ice.
3. Dilute 1ml of thawed Matrigel® in 4ml of DMEM/F12 in the 50ml tube.
4. Place the plates to be coated on ice and immediately distribute 4-5ml of
Matrigel® solution on 1 plate. Cover the surface completely and draw the
Matrigel® back into the cold pipette and quickly proceed to the next plate.
Diagram for 1:5 coating:
5. Repeat step 4 for two more cycles across all plates.

Be very careful not to create bubbles. If bubbles form, remove them with the pipette and recoat the plate.
6. Once all plates have been coated three times, aspirate off ALL excess Matrigel®.
7. Remove plates from ice; dry the outside of the plates on a clean paper towel in the hood.
8. Being careful to not let the plates dry out, immediately place cells onto the freshly coated plate. If the cell suspension has not been prepared, place RPE media onto plates and place in incubator until ready to use. When ready, aspirate the media and replace with the cell suspension.
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This procedure is recommended for 96 well, 12 well, and 24 well multi-well plates and permeable supports such as Transwell® and MilliCell® cell culture inserts.
*Note: Matrigel® is temperature sensitive and forms a gel when warmed to room temperature. To achieve optimal coating, work quickly, keep plates and reagents on ice, and use chilled pipettes and pipette tips to keep the Matrigel® cool.
1. Thaw one aliquot of Matrigel® overnight at 4°C. Put all plates and pipettes to be used in refrigerator at the same time to cool down to 4°C.If using coverslips, place the coverslips in an uncoated tissue culture dish and then cool.
2. Place 25ml of DMEM/F12 in a 50ml conical tube and place on ice.
3. Add 400 L of Matrigel® to the 50ml tube and gently mix.
4. Place the plates to be used on ice and immediately distribute Matrigel® solution on to the plates (100 μ L for 96 well plates, 0.5 ml on each ~1 cm diameter permeable support, enough to cover the surface of a coverslip, without coating the dish).
5. Place plates with Matrigel® in tissue culture incubator for 40 minutes
6. After 40 minutes, remove from incubator and aspirate all Matrigel®
7. Plate cells immediately or put media on the plates until ready to plate cells so the
Matrigel® has no chance to dry out. Plates with media on them can remain in incubator until ready to use.
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