1. Cancer and Fibroblast CHUNGNAM NATIONAL UNIVERSITY
MEDICAL SCHOOL of Biochemistry
JI Lab.
Hong Bum Park

2. Cancer association other cell
Fibroblast
BMSC
Tissue
MSC
Adipocyte

3. Cancer cell culture & heterogeneity
Cancer cell heterogeneity

4. Cancer cell by heterogeneity
Cancer cell by NGS
Cancer cell by RNA sequencing
Cancer cell by STR
Cancer cell culture condition
Animal model

5. Immunocytochemistry test
Nucleus  Dapi
Cytosol  rhodamine phalloidin
Cytokeratin(Dako)  FITC
CDD-18CO
DMEM
BMSC
DMEM
BMSC
IMDM
NHBF
DMEM
Primary Stromal Cell Line
Condition
BMSC : IMDM media, 10% FBS, 1% Penicilin-streptomycin-glutamine
CCD-18CO : DMEM, 10% FBS, 1% Penicilin-streptomycin-glutamine
NHFB : DMEM, 10% FBS, 1% Penicilin-streptomycin-glutamine
T47D : DMEM, 10% FBS, 1% Penicilin-streptomycin-glutamine
MDA-MB-231 : : DMEM, 10% FBS, 1% Penicilin-streptomycin-glutamine

6. Immunocytochemistry test
ab34710
anti-collagen 1 antibody 1
₩567,000
ab7778
anti-collagen 3 antibody
ab5694
anti-alpha smooth musle actin
ab28028
anti-vimentin
₩532,000
a0082
anti-factor 8 2ml
₩478,000
anti-factor ml
₩335,000
M0746
anti-HLA-DR
₩529,000

7. IMMUNOCYTOCHEMISTRY (ICC)
Fixation:
1. Fix the samples 3-4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature.
2. Wash the samples twice with ice cold PBS.
Permeabilization:
If the target protein is expressed intracellularly, it is very important to permeabilize the cells.
Note: acetone fixed samples do not require permeabilization.
3. Incubate the samples for 10 min with PBS containing 0.25% Triton X-100.
Triton X-100 is the most popular detergent for improving the penetration of the antibody. However,
it is not appropriate for the use of membrane-associated antigens since it destroys membranes.
4. Wash cells in PBS three times for 5 min.
Blocking and Incubation:
5. Incubate cells with 1% BSA in PBST for 30 min to block unspecific binding of the antibodies.
6. Incubate cells in the diluted antibody in 1% BSA in PBST in a humidified chamber for overnight at 4°C.
7. Decant the solution and wash the cells three times in PBS, 5 min each wash.
8. Incubate cells with the secondary antibody in 1% BSA for 1 hr at room temperature in dark.
9. Decant the secondary antibody solution and wash three times with PBS for 5 min each in dark.
Counter staining:
10. Incubate cells on μg/ml Hoechst or DAPI (DNA stain) for 1 min.
11. Rinse with PBS.
Mounting:
12. Mount coverslip with a drop of mounting medium.
13. Seal coverslip with nail polish to prevent drying and movement under microscope.
14. Store in dark at -20 or 4°C.

8. Immunocytochemistry : Cytokeratin.
A. T47D
B. MDA-MB-231
Dapi (Dako) 1:1000
Rhodamine phalloidin (invitrogen) 1 : 40
Cytokeratin (Dako, M3515) 1:100
Secondary antibody (invitrogen, A11029) 1: 500
Mount solution (Dako S3023)
Green: Cytokeratin
Red: Rhodamine Phalloidine
Blu: DAPI
C. Colon Fibroblast
D. Skin Fibroblast
E. BMSC
A. T47D, B MDA-MB-231, A and B is positive control, C. Colon Fibroblast, D. Skin Fibroblast, E. BMSC, C, D and E is negative control, Blue is nucleic, Red is actin, Green is Cytokeratin. X400, NIKON

9. Immunocytochemistry : Cytokeratin BMSC(P12) in DMEM
X400
X800 A. B. E. F. G. H. C. D.
Green: Cytokeratin
Red: Rhodamine Phalloidine
Blu: DAPI

10. Immunocytochemistry : Cytokeratin BMSC(P12) in IMDM
X400
X800 A. B. E. F. G. H. C. D.
Green: Cytokeratin
Red: Rhodamine Phalloidine
Blu: DAPI

11. Immunocytochemistry : Cytokeratin NHFB (P25, skin fibroblast)
X400
X800 A. B. E. F. G. H. C. D.
Green: Cytokeratin
Red: Rhodamine Phalloidine
Blu: DAPI

12. Immunocytochemistry : Cytokeratin CCD-18CO (P41, colon fibroblast)
X400
X800 A. B. E. F. G. H. C. D.
Green: Cytokeratin
Red: Rhodamine Phalloidine
Blu: DAPI

13. Immunocytochemistry : Cytokeratin MDA-MB-231 –breast cancer
X400
X800 A. B. E. F. G. H. C. D.
Green: Cytokeratin
Red: Rhodamine Phalloidine
Blu: DAPI

14. Immunocytochemistry : Cytokeratin T47D –breast cancer
X400
X800 A. B. E. F. G. H. C. D.
Green: Cytokeratin
Red: Rhodamine Phalloidine
Blu: DAPI

15. CCK-8 assay : Cell Proliferation Assay
Specific am : Establishment of mitomycin C Tx. Condition on feeder cell for growth arrest
Mitomycin C
5’-CpG-3’ sequence specific crosslinker
Day -1 Feeder cell seeding
D0 Mitomycin C Fx (4hr)
D3 CCK-8 assay
D6 CCK-8 assay
Cell count : 1000 cells/well
Cell line :
Skin fibroblast
BMSC
Cell seeding plan
Mitomycin C CN ug/ml
NHFB
BMSC
Culture Condition
DMEM 10% FBS, G-p/s 1%

16. CCK-8 assay : Cell Proliferation Assay
Specific am : Establishment of mitomycin C Tx. Condition on feeder cell for growth arrest
Mitomycin C
5’-CpG-3’ sequence specific crosslinker
Day -1 Feeder cell seeding
D0 Mitomycin C Fx (4hr)
D3 CCK-8 assay
D6 CCK-8 assay
Cell count : 1000 cells/well
Cell line :
Skin fibroblast
BMSC
CCK assay at Day 3

17. Protocol setup for colony pick-up
(Use of clonal ring)
Cell count : none
Cell line :
Skin fibroblast
MSC
MDA-MB-231
MCF 7
D-2 Feeder cell seeding
D-7 Colony pick-up (8*8)
D-0 Cancer cell seeding
D-1 Mitomycin C Fx (4hr)
cocktail
MDA-MB-231
MCF 7
NHFB & cocktail
MSC & cocktail

18. Co-culture (before colony pick-up)
A. . Skin fibroblast and cocktail
B. MSC and cocktail
C. MSC and cocktail
D. cocktail(MDA-MB-231, MCF 7)
E. MDA-MB-231
F. MCF 7
A. Skin fibroblast and cocktail B, C. MSC and cocktail D. cocktail is MDA-MB-231 and MCF 7 E. MDA-MB-231 F. MCF 7

19. Cells picked-up with clonal ring
A. . Skin fibroblast and cocktail
B. MSC and cocktail
C. MSC and cocktail
D. cocktail(MDA-MB-231, MCF 7)
E. MDA-MB-231
F. MCF 7
A. Skin fibroblast and cocktail B, C. MSC and cocktail D. cocktail is MDA-MB-231 and MCF 7 E. MDA-MB-231 F. MCF 7

20. Plan for colonal expansion of cancer cells
D-2 Feeder cell seeding
D-7 Colony pick-up (8*8)
D-0 Cancer cell seeding
D-1 Mitomycin C Fx (4hr)
Cell count :
Stromal cell : cell/well
Cancer cell : 100 cell/well
Cell line :
Skin fibroblast
MSC
MCF 7
MSC & MCF 7
NHFB & MCF 7
NHFB & MCF 7