MTT, Proliferation and Cytotoxicity Assay http://www.protocol-online.org/forums Fri, 10 Feb 2012 13:39:38 +0000 1000 Viability assay with propidium Iodide and Alamar Blue http://www.protocol-online.org/forums/topic/24375-viability-assay-with-propidium-iodide-and-alamar-blue/
I performed cell viabilitty assay using Propidium iodide and Alamar Blue. The experiment was performed using TECAN microplate reader.
The results I got as a RFU (relative fluorescence units). Now I want to calculate EC50, but I don't know how. I do not want use any software, can I use Excel? Does 4 different concentration is enough to calculate EC50? should this concentration be converted to log.?
And what about RFU values (axis -y), should I convert them as well?

I would be very grateful for any help.

Cheers,

Magda.]]> Fri, 10 Feb 2012 13:39:38 +0000 http://www.protocol-online.org/forums/topic/24375-viability-assay-with-propidium-iodide-and-alamar-blue/ MTT problems (Seeking advice) http://www.protocol-online.org/forums/topic/24042-mtt-problems-seeking-advice/ At first I am very grateful for every one who will read this topic
and deeply indebted for those who will share their experience, thoughts with me Posted Image .

Today was my first MTT assay on Tongue squamous cell carcinoma
I used a cytotoxic agent which is ZnCl2 to establish standard curve.
I used 10 microliter of the following concentration :
2 mg/ml
1mg/ml
0.5 mg/ml
0.1 mg/ml
0.01 mg/ml
0,001 mg/ml
0.0001 mg/ml
I followed the protocol of MTT assay as widely present online

what was strange that higher concentration showed a stronger purple color ( higher viability).
While low concentration showed a lower absorbance.

I wonder if any one could share his experience regarding using standard cytotoxic agents ( Not drugs ).
Best Regards]]> Tue, 17 Jan 2012 08:06:04 +0000 http://www.protocol-online.org/forums/topic/24042-mtt-problems-seeking-advice/ Performing MTT assay in suspension cells (new in the subject) http://www.protocol-online.org/forums/topic/23719-performing-mtt-assay-in-suspension-cells-new-in-the-subject/
I'm planning on using the MTT assay to evaluate the viability of my cells (THP-1 and PBMC) after treatment with some compounds. I've never performed this assay before so I collected some protocols but I'm still very confuse, so I would appreciate if someone could answer me a few questions:

1- My cells are in suspension. I don't know if I should treat them like that or if I should try to attach them by making a previous incubation. The problem is I don't know if they attach at all or if their behavior changes if they become adherent. On the other hand if I use them in suspension how am I suppose to remove the media before I add the MTT solution? Some refer a centrifugation step but I don't know how this can be done in a 96 well plate!

2- Some refer removing the media and wash after treatment with the compound and with the MTT solution while others don't. Are these steps necessary? (again the centrifugation problem!!!!!)

3- To dissolve the crystals, some use DMSO, others isopropyl alcohol/HCl, and others SDS/HCl. I don't know which solvent to choose.

(If an answer come from someone who works with my cell type please refer it)

Thanks very much in advance.]]> Fri, 09 Dec 2011 12:03:39 +0000 http://www.protocol-online.org/forums/topic/23719-performing-mtt-assay-in-suspension-cells-new-in-the-subject/ Contamination in human fibroblast culture http://www.protocol-online.org/forums/topic/23666-contamination-in-human-fibroblast-culture/
I am trying to grow primary fibroblasts from skin of sclreoderma patients. Since the very beginning, I have been observing tiny oval structures in bunches (like grapes) contaminating my cultures. This has also resulted in increased TNF levels.

I suspect this to be yeast contamination, I am not sure.

Please tell me:
  • How to ensure what kind of contamination is this?
  • Assuming that it is yeast contamination, how to get rid of it?
I am aware that I have given very limited information regarding my predicament, but I was unsure of what more to add. Please help me out, I'll be obliged

Regards
Maverick]]> Mon, 05 Dec 2011 12:09:23 +0000 http://www.protocol-online.org/forums/topic/23666-contamination-in-human-fibroblast-culture/ lymphocyte proliferation using trypan blue http://www.protocol-online.org/forums/topic/23636-lymphocyte-proliferation-using-trypan-blue/ I can not afford to buy an ELISA reader and I would like to test my lymphocyte proliferation using trypan blue. Is what I can do. Thu, 01 Dec 2011 14:37:35 +0000 http://www.protocol-online.org/forums/topic/23636-lymphocyte-proliferation-using-trypan-blue/ Measuring Calcium in the ER with mag fura 2 http://www.protocol-online.org/forums/topic/23542-measuring-calcium-in-the-er-with-mag-fura-2/ We are interested in determing if hypoxia/glucose deprivation cause release of calcium from the ER. WE plan to use mag fura2 to measure ER calcium. Two questions I have is there a way to measure calcium in real time, meaning being able to quench the reaction before taking it out of hypoxia, because reoxygenation can have an effect on calcium levels, and how do you harvest the cell after labeling. Thu, 24 Nov 2011 02:16:21 +0000 http://www.protocol-online.org/forums/topic/23542-measuring-calcium-in-the-er-with-mag-fura-2/ cyQuant for DNA content analysis http://www.protocol-online.org/forums/topic/23365-cyquant-for-dna-content-analysis/ thank you so much

Attached File(s)

]]> Wed, 09 Nov 2011 09:40:26 +0000 http://www.protocol-online.org/forums/topic/23365-cyquant-for-dna-content-analysis/ antibiotics interefere with MTT?? http://www.protocol-online.org/forums/topic/23124-antibiotics-interefere-with-mtt/
I'd like to know if antibiotics (Pen-Strep) interfere with MTT. I usually use medium without antibiotics but one of my friends followed the exact same protocol that I've been using so far, and for which I was getting good results, except she used medium with antibiotics. now even the control cultures aren't showing formation of formazan crystals after 4hrs of incubation.

the MTT stock was about 2weeks old so i tested it against a bacterial culture to see if that might be the problem, but the bacterial culture turned blue almost immediately.

So as I see it, the antibiotics could be the only problem. Is that right? can anyone provide me with some papers that document this [or the lack of interference]?

Thanks!]]> Mon, 17 Oct 2011 15:27:39 +0000 http://www.protocol-online.org/forums/topic/23124-antibiotics-interefere-with-mtt/ determining optimal cell count for MTT http://www.protocol-online.org/forums/topic/22866-determining-optimal-cell-count-for-mtt/
I'm new to the MTT assay and from what I've read, I have to first determine the optimal cell count before performing the assay with my test sample. None of the protocols that I've read for this [inlcuding the ATCC protocol attached], ask you to add any cytotoxic compound before adding the MTT. How then will the formazan crystals be formed if I'm going to plate cells with >90% viability? Or am I missing out on something?

Thanks]]> Wed, 21 Sep 2011 07:39:03 +0000 http://www.protocol-online.org/forums/topic/22866-determining-optimal-cell-count-for-mtt/ CFSE assay data analysis http://www.protocol-online.org/forums/topic/22799-cfse-assay-data-analysis/ I have the dot blot, need to analyze the proliferation]]> Thu, 15 Sep 2011 03:48:55 +0000 http://www.protocol-online.org/forums/topic/22799-cfse-assay-data-analysis/