Mast Cell adherence for CONFOCAL

[Diego A Goyeneche Patiño]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Currently I´m performing experiments with mast cells for Confocal Microscopy.
I adhere cells to commercial Poly Lysine coated coverslips however after fixation (PFA 2 % in PBS) and permeabilization (0,02 % Triton X 100)  I lose 70 % of the cells.
I used 5 x 105 - 1 x 106 cell to obtain at the end a decent number of cells. Since mast cells ( bone marrow derived) are difficult to grow and have them in a good number, I would like to ask an alternative protocol for cell adherence.

I want to mention that I have also coated my own glass and Plastic (thermanox) coverslips with Poly Lysine (1:100 and 1:1000 from stcok 0.1 % W/V) but I obtain the same result.

Is it true that  high concentrations of (1:10) Poly Lysine enhance background?

Really I would appreciate any suggestion in this matter!!

Diego A. Goyeneche Patiño 

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[Sylvie Le Guyader-2]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Diego

 

If your cells are well attached before fixation then the fixative or the handling are likely to be the problem. If they are not well attached, it is important not to shock them. I use N1E-115 cells which can be detached only by pipetting (no trypsin). I have no problem with fixation. This is what I would recommend:

-          make sure your cells are happy (fresh medium a few hours before, not confluent, warm…) before fixation

-          use reasonably new fixative only made from powder (as PFA/FA evaporates so ready-made stocks are not as good) as a 2x stock (4% in your case), aliquoted and frozen. Thawed aliquots can stay in the fridge for several weeks. Frozen aliquots are fine for a couple of years to my experience.

-          Prewarm the necessary volume of 2x fix to 37º

-          remove half of the medium from your cells (typically I grow my cells on 12mm diameter coverslips in 24-well plates with 500ul of medium so I remove 250 before fixation)

-          add the same volume of warm 2x fixative

-          return to the incubator for 5min

-          have a look again to see if cells are detached

Be quick with all these. Sensitive cells sometimes detach very fast when shocked for temperature or pH.

Good luck

 

Med vänlig hälsning / Best regards

 

Sylvie

 

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Sylvie Le Guyader

Dept of Biosciences and Nutrition

Karolinska Institutet

Novum

14157 Huddinge

Sweden

[phone]

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Diego A Goyeneche Patiño
Sent: 14 August 2008 16:45
To: [hidden email]
Subject: Mast Cell adherence for CONFOCAL

 

Currently I´m performing experiments with mast cells for Confocal Microscopy.
I adhere cells to commercial Poly Lysine coated coverslips however after fixation (PFA 2 % in PBS) and permeabilization (0,02 % Triton X 100)  I lose 70 % of the cells.
I used 5 x 105 - 1 x 106 cell to obtain at the end a decent number of cells. Since mast cells ( bone marrow derived) are difficult to grow and have them in a good number, I would like to ask an alternative protocol for cell adherence.

I want to mention that I have also coated my own glass and Plastic (thermanox) coverslips with Poly Lysine (1:100 and 1:1000 from stcok 0.1 % W/V) but I obtain the same result.

Is it true that  high concentrations of (1:10) Poly Lysine enhance background?

Really I would appreciate any suggestion in this matter!!

 

Diego A. Goyeneche Patiño 

 

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