Dear Dewi,

Generally, it is advised to make the medium  semi-solid by the addition of 0.5% Methylcellulose (R&D Systems, Minneapolis, MN) to prevent cell aggregation.

For details see:

Mammosphere Formation in Breast Carcinoma Cell Lines Depends upon Expression of E-cadherin

Juan Manuel Iglesias, Izaskun Beloqui, Francisco Garcia-Garcia, Olatz Leis, Alejandro Vazquez-Martin, Arrate Eguiara, Silvia Cufi, Andres Pavon, Javier A. Menendez, Joaquin Dopazo, Angel G. Martin

Published: October 4, 2013DOI: 10.1371/journal.pone.0077281

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077281

Abstract

Tumors are heterogeneous at the cellular level where the ability to maintain tumor growth resides in discrete cell populations. Floating sphere-forming assays are broadly used to test stem cell activity in tissues, tumors and cell lines. Spheroids are originated from a small population of cells with stem cell features able to grow in suspension culture and behaving as tumorigenic in mice. We tested the ability of eleven common breast cancer cell lines representing the major breast cancer subtypes to grow as mammospheres, measuring the ability to maintain cell viability upon serial non-adherent passage. Only MCF7, T47D, BT474, MDA-MB-436 and JIMT1 were successfully propagated as long-term mammosphere cultures, measured as the increase in the number of viable cells upon serial non-adherent passages. Other cell lines tested (SKBR3, MDA-MB-231, MDA-MB-468 and MDA-MB-435) formed cell clumps that can be disaggregated mechanically, but cell viability drops dramatically on their second passage. HCC1937 and HCC1569 cells formed typical mammospheres, although they could not be propagated as long-term mammosphere cultures. All the sphere forming lines but MDA-MB-436 express E-cadherin on their surface. Knock down of E-cadherin expression in MCF-7 cells abrogated its ability to grow as mammospheres, while re-expression of E-cadherin in SKBR3 cells allow them to form mammospheres. Therefore, the mammosphere assay is suitable to reveal stem like features in breast cancer cell lines that express E-cadherin.

Materials and Methods

Cell lines and culture conditions

MCF-7, T47D, MDA-MB-231, MDA-MB-435, MDA-MB-468 breast carcinoma cell lines was obtained directly from the ATCC (Manassas, VA, USA), JIMT-1 cells were obtained from the German Collection of Microorganisms, MDA-MB-436 and HCC-1937 were obtained from T. Stein (U. of Glasgow, UK, previously obtained from ATCC, Manassas, VA, USA) and SKBR3, BT474 and HCC-1569 were obtained form A. Pandiella (CIC-Salamanca, Spain, previously obtained from ATCC, Manassas, VA, USA) and grown in DMEM (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO) at 37°C in a 5% CO2incubator. Characterization of cell lines according to their surface expression phenotype can be found in Figure S1. For mammosphere formation, single cell suspensions were plated in 6-well tissue culture plates covered with poly-2-hydroxyethyl-methacrylate (Sigma, St. Louis, MO) to prevent cell attachment, at a density of 1,000 cells/ml in serum-free DMEM supplemented with 1% L-glutamine, 1% penicillin/streptomycin, 30% F12 (Sigma), 2% B27 (Invitrogen, Carlsbad, CA), 20 ng/ml EGF (Sigma, St. Louis, MO) and 20 ng/ml FGFb (Invitrogen, Carlsbad, CA). The medium was made semi-solid by the addition of 0.5% Methylcellulose (R&D Systems, Minneapolis, MN) to prevent cell aggregation. After 7 days in culture, mammospheres were collected by gentle centrifugation (200 x g) and dissociated enzymatically (5 min in 1:1 trypsin/DMEM solution at 37°C) and mechanically by passing through a 25G needle (6 strokes). Single cells were re-plated at a density of 1,000 cells/ml for subsequent passages.

Immunofluorescence

Mammospheres were collected by centrifugation in a Cytospin (Thermo, Waltham, MA, USA) onto glass slides prior to fixation. For monolayer cultures, cells were grown on LAB-TEK II chamber slides (Nalgene Nunc International, Penfield, NY). Cells were fixed in 4% paraformaldehyde for 15-30 min at room temperature, washed with PBS and permeabilized/blocked for 1h at room temperature with PBS, 0.3% Triton X-100, 2.5% horse serum. Slides were then incubated with anti-E-cadherin primary antibody (diluted 1:100) (seeFile S1) overnight, at 4°C washed in PBS. Appropriate fluorophore labeled secondary antibody was added at a dilution of 1/250 and incubated for 1 h at room temperature, and after washing in PBS, Hoescht 33342 dye was added to reveal nuclear DNA. Immunofluorescence was visualized in a Zeiss LSM510 confocal laser-scanning microscope.

Hoping this will be helpful,

Rafik

Hello DEWI, 

You might separate your cells to single cells by increasing the trypsinization time. If it is not efficient, your might use soft syringing(G25 syringe) to force cells to separate. Syringing might be harsh to cells if not applied adequately. Proceed FACS analysis assap to avoid additional cell death. 

Hope it helps. 

Best,

Hamadi. 

Try to include / increase EDTA-concentration in your sorting buffer - that should counteract E-cadherin mediated aggregation!

Good luck with your experiments,

Christian

thanks vey much for your information

hi

i had same problem, i have done any solution but still cells aggregate.

please help me!