I am working with LAD-2 cells (a human mast cell line).
I usually perform hemidepletion every 7 days. Within this period, I check the cells every 2-3 days and pipette up and down (with slow velocity, 10ml strippette) to dissociate cell clumps. Following my senior's protocol, I seed 2x10^6 cells into 10ml media (5ml old + 5ml new media) when I perform hemidepletion.
However, there are lots of cells clumps in my flasks. I do not know how to prevent cells from clumping.
I greatly appreciate any suggestion about LAD2 cell culture-method.
Thanks in advance.
Dear Le,
Your methods with the LAD2 cells are the correct one. My experience with the LAD2 cells was they aggreated more when they divided faster and they became less response to the stimuli as well. When I first work with them 8 years ago, they double in number every two weeks so I would not see that many cell clumps in the culture. As the cells double in number faster, they form more clumps.
Yes, I also observed what you say.
My cells response very weakly to the stimuli. Could you please suggest me how can I keep the cell to response better to the stimuli? I would like to perform beta-hexosaminidase release test using LAD-2 cells.
Thank you very much!
Dear Le,
Ensure you use the early passages if its possible, and keep as many early passage stocks for future use to ensure reproducible data.
Dear Laurie,
although my cells grow slowly but they make lots of clump. Is there any reason or how to stop them making clumps?
thank you again.
Dear Le,
It is really not a big problems when they clump together in the cluture fask. Just dispersed them with the 10ml or 25ml pippitte. However, you need to centrifuge them down, washes and disperse and count them just before incubate with stimuli or drugs so you know work with the same number of cells.
Thank you Laurie,
Have you every performed beta-hexosaminidase test with LAD2 cells? I performed the test but IgE-sensitized cells just release 4-5% compared to total cell lysate...
Do you have nay experience to share?
Thank you again
Dear Le,
I performed beta-hexosamidase assay test with IgE sensitized LAD2 cells stimulated with anti-Ige. 4-5% of net release was the value that we got in our lab. You can use histamine assay (expensive) to see if you get more release.
Thank you Laurie,
When I reviewed the references from other studies, they could get >20% net release of b-hexosaminidase from LAD2 cells... I wondered how they can get that value.
Do you have any idea?
Thanks again.
Dear Le,
The response of lad2 cells through IgE dependent release machanism inversely proportional to passage (Clulture generate). I did have very good response of lad2 cells when sensitized with serum of hay fever allergic patients and stimulate them with grass pollen about 7 or 8 years ago but not now. I don't have the early passage of lad2 cell stock anymore.
Dear Laurie,
How much was the serum volume that you used to incubate with LAD2 cells?
Thank you.
Dear Le,
I used up to 10% of serum for sensitization of LAD2 cell but 1 % only for RBL cells in order not to cell detachment from the wells.
Dear Laurie Lau,
Thank you so much for your information.
I am so glad to know that you also tested with RBL cells. It is not working also in my hand (so sad).
May I ask you a favor? Could you please send me the protocol for beta-hexosaminidase release test for both LAD2 and RBL cells you used previously?
I am so thankful to your help.
Best,
Duy.
The protocol for beta hexoaminidase assay for both LAD2 and RBL cells is use p-Nitrophenyl-N-acetyl-beta-D-Gucosaaminaminde (SigmaN-9376) as substrate (1.3mg/ml in 0.1M Na2HPO4, adjust pH to 4.5 with 0.4M citric acids, store at -25 C at the dark. We use 30 ul of cell supernatant + 50ul of substrate solution and incubate at 37C for 1hr and stop with0.2M glycine solution. It is important to determine the number of cells in your challenge well or tube by lysis eg 2 million of cells and constructed a standards curve by serial dilutions so that you won't use too many or too little cell for your challenge. Good luck.
Thank you Laurie Lau,
I would like to know how you sensitized the cells.
I seeded the cells 1x10^5 cells into 24 well plate the night before sensitization.
I sensitized RBL with anti DNP-mouse IgE (1ug/ml) overnight and stimulate with DNP-HSA (Sigma) 100ng/ml. However, the release of beta-hexosaminidase was very low.
Could you please again tell me how you sensitized and stimulated the cells?
Thank you very much!
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