That, unless you know a SC marker for your particular tumour under study, I'm afraid may be quite difficult but not impossible. There is no such an ubiquitous protocol yet; you will find in the literature that CSCs from some cancers like gioblastomas, melanomas, colon cancer, pancreas and breast cancer have indeed been successfully enriched and in some cases even isolated. However, the approaches are quite different, some of them very particular, as it ofter if not always the success of such processes depends on the tumour type and also the initial abundance of the CSC in the tumour tissue or culture. It won't be the same to isolate hematopoietic cancer stem cells than Small Cell Lung Cancer stem cells (the later have not been isolated, only their existence was inferred). You definitely need markers that will allow you to identify and subsequently isolate CSCs in your cultures.

I have pasted a list (at bottom of the answer) including the most common/reported markers used to identify CSCs in various tumour types.

You need to find the right one or combinations of them that will aid you in the identification/enrichment/isolation. For example, in my experience with SCLC, CD33 wasn't a useful CSC marker as most SCLC cells in the bulk of cultures of tumours expressed it.

Some people advocate the following alternatively: chemo/radio therapy works against all rapidly dividing cells. But there is increasing evidence that cancer stem cells are more resistant than other cells to this treatment...so, targeting the bulk of your cultures may leave behind cells that are innately resistant (enrichment), than can self-renew and can eventually repopulate the tumour/culture (Recalling: the consensus definition of a cancer stem cell is a cell within a tumor/culture that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor).

Once you got this innately resistant population you could test the enrichment using some of the canonical stem cell surface markers (list) identified in other tumours and, subsequently obviously test the tumourigenicity of these potential CSC (a.k.a. tumour initiating cell) vs. the bulk of cells' (therapy sensitive).

The therapy, in theory, should have enriched your cultures in these innately resistant cells. During the "regeneration", that is relapse of your culture, these cells will divide to start repopulating the culture. Moreover, they will have to self-renew even before starting repopulating the culture. Therefore every time they divide (even they do it asymmetrically, that is, CSC--> CSC + transient amplifying) they'll generate one additional copy of themselves (this is just the theory)....so your cultures will become enriched after subsequent rounds of therapy (some chemoresistance literature support this theory).

An easier start point also could be the in vitro version of serial tumour transplantation, conventionally used as a surrogate marker of stem-ness in a tumour. You could perform a serial dilution assay to plate single cells and look for single-cell containing-wells that formed colonies (CFU, see below). This assay can also be used to enrich your cultures in CSC as every time you re-plate a colony the number of CSC in each colony will theoretically increase giving the self-renewal capacity of the CSC that initiate the colony in the first place.

An ideal in vitro assay would be (a) quantitative; (b) highly specific, measuring only the cells of interest; (c) sufficiently sensitive to measure candidate stem cells when present at low frequency; and (d) rapid. Several in vitro assays have been used to identify stem cells, including sphere assays, serial colony-forming unit (CFU) assays (replating assays), and label-retention assays.

Good Luck

List:

Bladder Cancer Stem Cell Markers

CD44

CD47

Breast Cancer Stem Cell Markers

Aldehyde Dehydrogenase 1-A1/ALDH1A1

GLI-1

BMI-1

GLI-2

CD24

IL-1 alpha/IL-1F1

CD44

IL-6 R alpha

CXCR4

CXCR1/IL-8 RA

DLL4

Integrin alpha 6/CD49f

EpCAM/TROP1

PTEN

ErbB2/Her2

Colon Cancer Stem Cell Markers

ALCAM/CD166

EpCAM/TROP1

Aldehyde Dehydrogenase 1-A1/ALDH1A1

GLI-1

CD44

Musashi-1

DPPIV/CD26

Gastric Cancer Stem Cell Markers

CD44

DLL4

Glioma/Medulloblastoma Cancer Stem Cell Markers

A20/TNFAIP3

Integrin alpha 6/CD49f

ABCG2

L1CAM

CX3CL1/Fractalkine

Musashi-1

CX3CR1

c-Myc

CXCR4

Nestin

HIF-2 alpha/EPAS1

Podoplanin

IL-6 R alpha

Head & Neck Cancer Stem Cell Markers

ABCG2

BMI-1

Aldehyde Dehydrogenase 1-A1/ALDH1A1

CD44

Leukemia Cancer Stem Cell Markers

BMI-1

GLI-2

CD34

IL-3 R alpha

CD38

MICL/CLEC12A

CD44

Musashi-2

CD47

SCF R/c-kit

CD96

TIM-3

GLI-1

Liver Cancer Stem Cell Markers

alpha-Fetoprotein/AFP

CD90/Thy1

Aminopeptidase N/ANPEP

NF2/Merlin

Lung Cancer Stem Cell Markers

ABCG2

EpCAM/TROP1

Aldehyde Dehydrogenase 1-A1/ALDH1A1

SCF R/c-kit

CD90/Thy1

Melanoma Cancer Stem Cell Markers

ABCB5

MS4A1/CD20

ABCG2

Nestin

ALCAM/CD166

NGF R/TNFRSF16

Myeloma Cancer Stem Cell Markers

CD19

MS4A1/CD20

CD27/TNFRSF7

Syndecan-1/CD138

CD38

Osteosarcoma Cancer Stem Cell Markers

ABCG2

STRO-1

Nestin

Ovarian Cancer Stem Cell Markers

alpha-Methylacyl-CoA Racemase/AMACR

SCF R/c-kit

CD44

Pancreatic Cancer Stem Cell Markers

BMI-1

CXCR4

CD24

EpCAM/TROP1

CD44

Prostate Cancer Stem Cell Markers

ABCG2

CD44

alpha-Methylacyl-CoA Racemase/AMACR

c-Myc

That, unless you know a SC marker for your particular tumour under study, I'm afraid may be quite difficult but not impossible. There is no such an ubiquitous protocol yet; you will find in the literature that CSCs from some cancers like gioblastomas, melanomas, colon cancer, pancreas and breast cancer have indeed been successfully enriched and in some cases even isolated. However, the approaches are quite different, some of them very particular, as it ofter if not always the success of such processes depends on the tumour type and also the initial abundance of the CSC in the tumour tissue or culture. It won't be the same to isolate hematopoietic cancer stem cells than Small Cell Lung Cancer stem cells (the later have not been isolated, only their existence was inferred). You definitely need markers that will allow you to identify and subsequently isolate CSCs in your cultures.

I have pasted a list (at bottom of the answer) including the most common/reported markers used to identify CSCs in various tumour types.

You need to find the right one or combinations of them that will aid you in the identification/enrichment/isolation. For example, in my experience with SCLC, CD33 wasn't a useful CSC marker as most SCLC cells in the bulk of cultures of tumours expressed it.

Some people advocate the following alternatively: chemo/radio therapy works against all rapidly dividing cells. But there is increasing evidence that cancer stem cells are more resistant than other cells to this treatment...so, targeting the bulk of your cultures may leave behind cells that are innately resistant (enrichment), than can self-renew and can eventually repopulate the tumour/culture (Recalling: the consensus definition of a cancer stem cell is a cell within a tumor/culture that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor).

Once you got this innately resistant population you could test the enrichment using some of the canonical stem cell surface markers (list) identified in other tumours and, subsequently obviously test the tumourigenicity of these potential CSC (a.k.a. tumour initiating cell) vs. the bulk of cells' (therapy sensitive).

The therapy, in theory, should have enriched your cultures in these innately resistant cells. During the "regeneration", that is relapse of your culture, these cells will divide to start repopulating the culture. Moreover, they will have to self-renew even before starting repopulating the culture. Therefore every time they divide (even they do it asymmetrically, that is, CSC--> CSC + transient amplifying) they'll generate one additional copy of themselves (this is just the theory)....so your cultures will become enriched after subsequent rounds of therapy (some chemoresistance literature support this theory).

An easier start point also could be the in vitro version of serial tumour transplantation, conventionally used as a surrogate marker of stem-ness in a tumour. You could perform a serial dilution assay to plate single cells and look for single-cell containing-wells that formed colonies (CFU, see below). This assay can also be used to enrich your cultures in CSC as every time you re-plate a colony the number of CSC in each colony will theoretically increase giving the self-renewal capacity of the CSC that initiate the colony in the first place.

An ideal in vitro assay would be (a) quantitative; (b) highly specific, measuring only the cells of interest; (c) sufficiently sensitive to measure candidate stem cells when present at low frequency; and (d) rapid. Several in vitro assays have been used to identify stem cells, including sphere assays, serial colony-forming unit (CFU) assays (replating assays), and label-retention assays.

Good Luck

List:

Bladder Cancer Stem Cell Markers

CD44

CD47

Breast Cancer Stem Cell Markers

Aldehyde Dehydrogenase 1-A1/ALDH1A1

GLI-1

BMI-1

GLI-2

CD24

IL-1 alpha/IL-1F1

CD44

IL-6 R alpha

CXCR4

CXCR1/IL-8 RA

DLL4

Integrin alpha 6/CD49f

EpCAM/TROP1

PTEN

ErbB2/Her2

Colon Cancer Stem Cell Markers

ALCAM/CD166

EpCAM/TROP1

Aldehyde Dehydrogenase 1-A1/ALDH1A1

GLI-1

CD44

Musashi-1

DPPIV/CD26

Gastric Cancer Stem Cell Markers

CD44

DLL4

Glioma/Medulloblastoma Cancer Stem Cell Markers

A20/TNFAIP3

Integrin alpha 6/CD49f

ABCG2

L1CAM

CX3CL1/Fractalkine

Musashi-1

CX3CR1

c-Myc

CXCR4

Nestin

HIF-2 alpha/EPAS1

Podoplanin

IL-6 R alpha

Head & Neck Cancer Stem Cell Markers

ABCG2

BMI-1

Aldehyde Dehydrogenase 1-A1/ALDH1A1

CD44

Leukemia Cancer Stem Cell Markers

BMI-1

GLI-2

CD34

IL-3 R alpha

CD38

MICL/CLEC12A

CD44

Musashi-2

CD47

SCF R/c-kit

CD96

TIM-3

GLI-1

Liver Cancer Stem Cell Markers

alpha-Fetoprotein/AFP

CD90/Thy1

Aminopeptidase N/ANPEP

NF2/Merlin

Lung Cancer Stem Cell Markers

ABCG2

EpCAM/TROP1

Aldehyde Dehydrogenase 1-A1/ALDH1A1

SCF R/c-kit

CD90/Thy1

Melanoma Cancer Stem Cell Markers

ABCB5

MS4A1/CD20

ABCG2

Nestin

ALCAM/CD166

NGF R/TNFRSF16

Myeloma Cancer Stem Cell Markers

CD19

MS4A1/CD20

CD27/TNFRSF7

Syndecan-1/CD138

CD38

Osteosarcoma Cancer Stem Cell Markers

ABCG2

STRO-1

Nestin

Ovarian Cancer Stem Cell Markers

alpha-Methylacyl-CoA Racemase/AMACR

SCF R/c-kit

CD44

Pancreatic Cancer Stem Cell Markers

BMI-1

CXCR4

CD24

EpCAM/TROP1

CD44

Prostate Cancer Stem Cell Markers

ABCG2

CD44

alpha-Methylacyl-CoA Racemase/AMACR

c-Myc

You could try using the anti-CD44v9 antibodies which seems to play an important roll in several cancer cells.

References:

http://www.ncbi.nlm.nih.gov/pubmed/23778530

http://www.ncbi.nlm.nih.gov/pubmed/23800986

anti-CD44v9 antibodies suppliers:

http://search.cosmobio.co.jp/cosmo_search_p/search_gate2/docs/CAC_/LKGM001.20130626.pdf

http://www.linscottsdirectory.com/antibodies/american-type-culture-collection/cd44-v9-variant-human-2655188

Enriching and increasing the tumorigenic potential of cancer stem cells can be done by serial passaging cell lines in mice models (see the paper link and the references within)

https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis

Note: the cell line should be from solid tumor types not from hematopoietic cancers, so that the tumor is localized (such as sub-cute injections or orthotopic injections) and will be easy to take it back to in vitro systems. Note the time taken for tumor formation at each passage in vivo, it should form tumors rapidly when you reach higher passage numbers. Choose markers based on the tumor type your cell line(s) belongs to. If the cell line belongs to male/female choose the mice male/female accordingly (to have the hormonal control in vivo). Some cancers such as breast cancer cells are highly influenced by estrogen. Good luck..!!

Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...

Some useful info can be found here:

http://www.sigmaaldrich.com/technical-documents/articles/biofiles/the-cancer-stem-cell.html