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Comparative Physiology @ Guelph

Keratinocyte Media and Culture Protocols

3 parts DMEM+ 1 part HAMS F-12

Supplements:

1. Adenine (MW=184.2)
663 mg in 20ml sterile dH2O.
Increase pH with NaOH to dissolve
–    add 18ml d H2O then NaOH drop-wise (10M NaOH i.e. 4g in 10ml)
–    Make to Vf=20ml
2. Hydrocortisone
8mg in 20ml sterile dH2O
3. Transferrin
100mg in 20ml sterile dH2O
4. Lyothyronine (MW=673)
-1mg dissolved in 1ml NaOH
-Swirl to dissolve 
-Add 49 ml dH2O
Serial dilute this 1:10-1:10-1:15 ([]f=2x10-18M)
5. Insulin
10 mg insulin + 2mL 0.005 N HCL
Store at 4 ºC (Max. 2 weeks)
0.5 mL in 500 mL medium 
* If necessary, warm gently in water bath to dissolve
6. EGF Human recombinant
200µg dissolved in 20ml sterile dH2O. []f=10µg/ml

All supplements then aliquoted to 1ml (EGF in 0.5ml) aliquots.
0.5ml of each supplement added to each 500ml of RM media.
Only RM+ contains EGF.

Make up RM bottles then filter sterilize. You can pour supplements in as you go. Do RM- first and then RM+. It is usually OK to do 1 bottle RM- and 1 RM + from the same filter.

CELLS

Keratinocytes were originally immortalized in presence of fibroblasts.  These were needed to make ECM cauparents. There are still fibroblasts in the cell lines and you need to keep on top of them.

Splitting cells (Based on 100% confluent cultures – adjust as needed):

1.    Aspirate off media of the cultured cells
2.    Wash with 5 ml of sterile 1x PBS solution & aspirate off
3.    Wash with 5 ml of sterile 1x PBS / EDTA solution & aspirate off
4.    Add 1 ml of 0.25% Trypsin:PBS and incubate cells for 10 minutes at 37 0C & 5 % CO2 until cells detach. This can take longer if cells are confluent. Sometimes banging the flask helps.
Re-splitting to tissue culture plate:
5.    Add 5 ml of 1x PBS to the 1 ml of Trypsin in the dish (6 ml total) and then pipette 1 ml of the solution of cells into each new culture plate. (if you have a lower confluent of cells use less PBS therefore concentrating the solution of cells)    
6.    Add 10 ml of RM- (pre-warmed) to the new culture plates to inactivate Trypsin. How much RM- you add depends on what you want to do with the cells.
7.    Next day, replace media from RM- to RM+ by aspirating off the old media and applying the new 10 ml of RM+ (RM+ = growth factor). RM- only used for splitting cells. You can however in cases of emergency (i.e. Friday afternoon) split them using RM+.
Splitting to small Petri-plates containing specifically coated membranes:
5.    Add 11 ml of 1x PBS to the 1 ml of Trypsin in the dish (12 ml total) and then              pipette 0.5 ml of the solution of cells into each new small culture plate with membrane (for 100% confluent cells, adjust as needed)
6.    Add 5 ml of RM- to each small culture plate to inactivate Trypsin & Next day switch media from RM- to 5 ml RM+

Stock solutions:

10x PBS

20g  NaCl
3.6g Na2HPO4
0.6g KH2PO4
0.5g KCl
Dissolve in 180 ml dH2O. pH to 7.4 then make up to final volume of 250 ml.

PBS / EDTA

MW=372.24
0.372g in 10ml=100mM
Add 1ml EDTA stock per 100 ml 1x PBS. [EDTA]f = 1 mM

Freezing cells down:

Freezing mix: 8% DMSO in FBS.
Trypsinise cells. Resuspend in 5 ml pre-warmed medium (without antibiotics). Centrifuge at 100 rpm for 10 minutes. Aspirate medium, then leave tube for a couple of minutes, then aspirate again. Resuspend pellet in 1ml freezing mix. Transfer to cryotube and put in -80oC.

Antibodies:

M u001( anti-k14) use @ 1:500
P BL-18(anti-k5) use @ 1:2000
P FAK use @ 1:250
M pFAK use @ 1:50

M LJ4 (anti-phospho-keratin) use @ 1:50

Desmosomal antibodies
All used @ 1:100

65160- anti-plakophilin
233- anti-BP180
11-5F- anti-desmoplakin
5q11-anti-Dsg3
1E5- anti-BP230

Cells in -80oC:
NEB-1 wt keratinocytes
KEB-7 carry R125P mutation
KEB-4 carry K14V270M (mild phenotype)