Cell Viability Assay

Cell Viability Assay

  • María Sánchez-Osuna added an answer:
    4
    Please how long does the staining stay on cells, can you still image after 24hrs and the stained cells will still retain their stain?

    The idea of fixing the cells with 4% Paraformaldehyde/ ethanol is to make sure the cells do not wash off and are still present during the whole process .
    So if I wanted to do this experiment the steps I would follow would be to

    1. Remove media in which cells are initially growing
    2. Wash gently with PBS without disrupting cells too much
    3. Fix with 4% PFA for 10mins at room temperature
    4. Incubate with mix of Calcien-AM and EtBr in PBS/serm free media for 30mins @ 37degrees

    Now my doubts are should i skip step 3 and just go directly to step 4 or what other steps do I need to include so my staining works perfectly.

    Please how long does the staining stay on cells, can you still image after 24hrs and the stained cells will still retain their stain?

    María Sánchez-Osuna

    Dear Hakeem, 

    I am not sure about what do you want to do with this protocol. Calcein AM is a cell-permeable dye mostly used to stain viable cells. Therefore, you can not fix your cells before staining them with calcelin AM. Since calcein AM need a viable cytosolic sterase for becoming fluorescent, I don't think you are able to see green if you previously fix your sample.

    In my opinion, if you want to see viable cells: steps 1, 2 and 4 (without 3). Nevertheless, there are other options to distinguish alive from dead cells besides calcein AM (just in case you have troubles with this protocol). If you want to see all cells from your experiment, used the entire protocol but only with EtBr, Hoechst, or PI in step 4, to visualize cell nuclei from all (alive and dead) cells. 

    Regards,

  • Xiarong Shi added an answer:
    7
    Strange results with TF-1 cell line and CellTiter-Glo Luminescent Cell Viability Assay
    My lab is experiencing unstable results with TF-1 cells and the CellTiter-Glo kit by Promega.
    It's a very easy assay, seeding different amount of cells, growing for 3 days, then measuring via CellTiter-Glo. Strange thing is, the results are not constant, meaning we have triplicates and e.g. 2 wells are about the same value, one is about 1% of the other values. There is not even one seeding-concentration with all values about the same level.

    On the other hand we tried MTT-assay. This showed very good and constant results! No extreme-low wells at all.

    It it possible that TF-1 cells are acting strange with CellTiter-Glo? I don't think that it's a seeding-problem, a mulitchannel-pipette was used. And why is MTT giving such good results? Are TF-1 cells sensitive to shaking? We used NFS60 and Raji cells before, with fine results. We shake 15min then incubate for 15min, is this too much for TF-1 cells?
    Xiarong Shi

    it sounds like to me that your plate is off the position in the reader, I am assuming you were using a microplate reader. do you find the odd reading wells in particular position in the plate? may be the edge may be certain row or column etc.

  • Roberta Ricciarelli added an answer:
    1
    How should SHSY5Y cells be treated with Beta Amyloid 25-35 that it can decrease their viability?

    I want use Beta amyloid 25-35 as a neurotoxin for SHSY5Y cells.Although in articles 10 microM of it for 24h is used and cell viability of them decreased approximately  20% but in my experiments with two bath from different company even in 200 microM of it  , there was no decrease in cell viability. my protocol is: Briefly, Aβ25–35 was initially dissolved in double-distilled water to 1 mM. The peptide solution was divided into aliquots and stored at −20°C. Before use, the Aβ25–35 solution was incubated at 37°C for 7–10 days to form aggregated diffusible oligomers, then diluted in medium to the indicated concentration. When I treated cells with BA i tested 10% and 2% FBS in media and both of them has the same result. what  is your recommendation?

    Roberta Ricciarelli

    My suggestion is to increase the amyloid concentration to 1 mM and, if it still doesn't work, move to the 1-42 peptide, at least as a positive control.

    SHSY5Y are cancer cells and, therefore, they may develop higher resistance to pathogenic stumuli. Primary neuronal cultures would be a better model.

  • Robert Kiss added an answer:
    1
    Can anyone advise on MTT cell proliferation assay?
    I have done MTT cell proliferation assay for H2O and MeOH extract of my plant sample using MCF7 cell line. I seeded 2000 cells/well in 96 well plate using phenol red free RPMI with 5% charcoal stripped FBS and the results were quite off and the absorbance at 550 nm was around 0.9-1.0 for triplicate. But when I did it with DCM extracts following the same method the absorbance was very low, around 0.2-0.5 for all concentration of extract and even the Growth control (cells only without treatment) and the Positive control (estradiol). I repeated it 2-3 times but am still having the same problem. Can anyone suggest me what to do? And why having this problem?
    Robert Kiss

    You should contact (on my behalf) my colleague Véronique Mathieu, MD, PhD at vemathie@ulb.ac.be

    I am sure that she will be able to help you.

    Best regards

    Robert

  • Dyaningtyas Dewi Pamungkas Putri added an answer:
    6
    Should I be seeing a yellow color after solubilizing formazan crystals in MTT assay?

    Hi everyone, 

    I am very new to MTT assay and I find myself stuck. 

    I plated 5,000 cells/well in a 96-well plate. Following recovery and incubation, I added 50uL MTT reagent to each well and incubated for 3 hours. 

    I did see formation of blue crystals in all wells. However, after removing the media and solubilizing with 0.04M HCl in isopropanol (200uL/well), I only see a yellow color-no purple. 

    Is there a reason why I would see yellow and not purple? Should I rinse the wells with PBS before solubilizing? 

    Thanks!

    Dyaningtyas Dewi Pamungkas Putri

    hi Elizabeth Stewart,

    As the others, I t might be your cells is not enough, death or low activity. But It is better before you treat with MTT check first your condition cells under microscope. you can put positive control too (e.g. untreated cells), so you can find the problem whether the amount of the cells, death cells or low activity. Actually you should put untreated cells to measure the activity of your sample. can you tell me about the concentration of your mtt?

    hope help you, good luck

  • Mansoureh Hashemi added an answer:
    14
    What is best solvent of MTT powder for cell viability assay?
    I want to assay cell viability with MTT powder from Merck company. What is best solvent of MTT powder for cell viability assay?
    Thanks.
    Mansoureh Hashemi

    it mix at slow speed on a magnetic stir plate for 15 minutes until the MTT powder is completely dissolved.

  • Kevin Stoltz added an answer:
    9
    How can I determine cell viability with fixed sections?

    I would like to examine the cell viability of my tissue-engineered constructs in regards to distribution. TUNEL staining is an option, but the kits are rather expensive.

    I've heard that it may be possible to do typical live/dead stain (i.e. calcien-AM, PI stains) prior to the sectioning. Have anyone tried this before and could you share your experiences with this method? My concern is the preparation step (i.e. washing, fixation, OCT infiltration timing, etc) after the staining and how these factors would affect the final fluorescence signal.

    Thanks in advance!

    Andy

    Kevin Stoltz

    Hi Andy,

    I am currently attempting something similar with a porcine scleral organ culture system. Did you have any luck using the alive/dead stains?

    I was planning to use ethidium homodimer, calcein AM and Hoechst prior to embedding in OCT and sectioning, while skipping fixation all together.  

    Thanks, 

    Kevin 

  • Rafik Karaman added an answer:
    1
    Can someone suggest viable methods of morphological analysis for HCT & HEK cells?

    We're currently studying the cytotoxic effects of a plant extract on HCT & HEK cells, and we want to incorporate a morphological analysis of our cells during the pre-introduction and post-introduction of the extract. Could anyone suggest literature or a method of analysis for this?

    Any help would be very much appreciated.

    Rafik Karaman

    Dear Jason,

    The following publications cover the answer to your question:

    1-Synthesis and biological evaluation of 6H-1-benzopyrano[4,3-b]
    quinolin-6-one derivatives as inhibitors of colon cancer cell growth
    Tie-Ling Li1
    , Hai-Feng Guo2, Fu-Juan Li3, Zhi-Guo Sun2 and Heng-Chun Zhang2

    Abstract:

    A convenient synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6-one derivatives
    was reported using 4-chloro-2-oxo-2H-chromene-3-carbaldehyde with different
    aromatic amines using silica sulfuric acid. The compounds were tested for
    their anticancer activity against colon (HCT-116 and S1-MI-80), prostate (PC3
    and DU-145), breast (MCF-7 and MDAMB-231) cancer cells. These compounds
    showed more selectivity and potent cytotoxic activity against colon
    cancer cells. 3c was tested against five other colon cancer cell lines (HT-29,
    HCT-15, LS-180, LS-174, and LoVo), which had similar cytotoxicity and
    selectivity. 3c did not induce PXR-regulated ABCB1 or ABCG2 transporters.
    In fact, 3c induced cytotoxicity in HEK293 cells over expressing ABCB1 or
    ABCG2 to the same extent as in normal HEK293 cells. It was cytotoxic
    approximately 3- and 5-fold to resistant colon carcinoma S1-MI-80 cells. 3c
    also produced concentration-dependent changes in HCT-116 colon cancer
    cells, in mitochondrial membrane potential, leading to apoptosis, and submicromolar concentrations caused chromosomal DNA fragmentation.

    Cell culture
    Colon carcinoma cell lines [HCT-116, HCT-15, HT-29, Lovo, LS-180, LS-174, S1 (a clone of LS174T cells)] and prostatic cancer cell lines [DU-145 and PC -3] and breast carcinoma cells [MDA-MB-231 and MCF-7] were grown as adherent monolayers in flasks with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator containing 5% CO2 at 37ºC. A G482 mutant ABCG2-overexpressing, drug-resistant colon cancer cell line, S1-M1-80, was maintained in medium with 80 µM mitoxantrone (Miri et al., 2011; Robey et al., 2001). The assay media for PXR transactivation assays included phenol red-free DMEM (Lonza) supplemented with
    5% charcoal/dextran-treated FBS (HyClone) and the other additives.
    PXR transactivation assay HepG2 cells were transfected with pcDNA3-hPXR
    and CYP3A4-luc plasmids using FuGENE 6 (Promega). After 24 hours of transfection in growth media, 10,000 cells were plated into wells of 96-well
    culture plates (PerkinElmer), and treated with DMSO, RIF, or 3c for an additional 24 hours. At 48 hours of transfection, a luciferase assay was performed to measure luminescence using the Neolite Reporter Gene Assay System (PerkinElmer) and a
    FLUOstar Optima microplate reader (BMG Labtech). Normalized CYP3A4 promoter activity was expressed as fold induction over the DMSO control. Cell viability was measured in parallel by CellTiter-Glo luminescent assays (Promega), which determine the number of metabolically active cells by quantifying the ATP present. Luminescence was measured with a FLUOstar Optima plate reader (BMG Labtech).
    Cell cytotoxicity as determined by MTT assays and morphological analysis
    The MTT assay (Carmichael et al., 1987) was used to determine cytotoxicity of the compounds to the following cells: HCT-116, HCT-15, HT-29, LS-180, LS-174,
    Lovo, S1, DU-145, PC-3, MDA-MB-231 and MCF-7. Briefly, the cells were harvested with trypsin and suspended at a final concentration of 5 X 103 cells/well. Cells were seeded (180 µL/well) into 96-well multiplates. Different concentrations of 6H-1-benzopyrano [4,3-b]quinolin-6-one derivatives (20 µL/well) were added. After 72 hours of incubation, 20 µL of MTT solution (4 mg/mL) was added in each well, and the plates were incubated further 4 hours, allowing the viable cells to convert the yellow-colored MTT into dark -blue formazan crystals. Subsequently, the medium was removed, and DMSO (100 µL) was added in each well to dissolve the formazan crystals. The absorbance was determined at 570 nm with an OPSYS microplate Reader (DYNEX Technologies, Inc., Chantilly, VA, USA). The means ± SD concentrations were calculated from at least three experiments performed in triplicate. The IC50 values were calculated from survival curves using the Bliss method. At 68 hours, cells with or without treatment were photographed by use of an inverted microscope (Olympus, BX53F) with fluorescent lamps and digital cameras. The data were acquired and analyzed by CellSens software.

    For more details, please see attached file.

    2-Bioorg Med Chem. 2015 May 1;23(9):2148-58. doi: 10.1016/j.bmc.2015.03.002. Epub 2015 Mar 21.
    Design, synthesis and in vitro cell-based evaluation of the anti-cancer activities of hispolon analogs.
    Balaji NV1, Ramani MV1, Viana AG2, Sanglard LP2, White J2, Mulabagal V3, Lee C2, Gana TJ4, Egiebor NO5, Subbaraju GV1, Tiwari AK6.
    Author information
     
    Abstract
    Phytochemicals play an important role in cancer therapy. Hispolon and 26 of its analogs (9 known and 17 new) were synthesized and evaluated for their antiproliferative activities in a panel of six independent human cancer cell lines using the in vitro cell-based MTT assay. Among the hispolon analogs tested, compound VA-2, the most potent overall, produced its most significant effect in the colon cancer cell lines HCT-116 (IC₅₀ 1.4 ± 1.3 μM) and S1 (IC₅₀ 1.8 ± 0.9 μM) compared to its activity in the normal HEK293/pcDNA3.1 cell line (IC₅₀ 15.8±3.7 μM; p<0.01 for each comparison). Based on our results, VA-2 was about 9- to 11-times more potent in colon cancer cells and 2- to 3-times more potent in prostate cancer cells compared to HEK293/pcDNA3.1 cells. Morphological analysis of VA-2 showed significant reduction of cell number, while the cells' sizes were also markedly increased and were obvious at 68 h of treatment with 1 μM in HCT-116 (colon) and PC-3 (prostate) cancer cells. A known analog, compound VA-4, prepared by simple modifications on the aromatic functional groups of hispolon, inhibited prostate and colon cancer cell lines with IC₅₀ values <10 μM. In addition, hispolon isoxazole and pyrazole analogs, VA-7 and VA-15 (known), respectively, have shown significant activity with the mean ICv values in the range 3.3-10.7 μM in all the cancer cell lines tested. Activity varied among the analogs in which aromatic functional groups and β-diketone functional groups are modified. But the activity of analogs VA-16 to VA-27 was completely lost when the side chain double-bond was hydrogenated indicating the crucial role of this functionality for anticancer activity. Furthermore, many of the compounds synthesized were not substrates for the ABCB1-transporter, the most common cause of multidrug resistance in anti-cancer drugs, suggesting they may be more effective anticancer agents.

    http://www.ncbi.nlm.nih.gov/pubmed/25842364

    Hoping this will be helpful,

    Rafik

  • Jolieke van Oosterwijk added an answer:
    4
    Has anybody had experience with MTT in non adherent cells?

    Has anybody had experience with MTT in non adherent cells?

    Jolieke van Oosterwijk

    Good Luck!

  • Paolo Bonaiuti added an answer:
    4
    How do I determine the viability of yeast on Nocodazole agar plates?

    I need to determine yeast viability upon exposure to Nocodazole. I need to prepare SD plates containing Nocodazole. I know it is hard to be dissolved but if someone has used it on plates ( if it is possible, I have seen Benomyl plates, but never NOC on plates)  it would be much appreciate to know what rank of concentrations should I use. Also if there is any recommendation in order to make the plates.

    One more question: how long are such plates stable: over a day? 

    If anyone has such an expertise that would be very helpful.

    Paolo Bonaiuti

    hi Nerea,
    I use W303 background strains, and 15ug/ml as final concentration is fine, starting from 1.5mg/ml solution in DMSO.
    this is quite the typical concentration.
    I think you could find some info on the papers from the '90s, where they screened for Spindle Assembly Checkpoint components (like Li and Murray, 1991).

    all the best,
    Paolo

  • Julio César Sánchez Pech asked a question:
    Open
    What is the protocol for LIVE/DEAD Cell Viability Assays of smooth muscle cell?

    I want to know what is the correct protocol, thanks in advance.

  • Parag Kolekar added an answer:
    4
    How do I calculate toxicity in the MTT assay?

    I am performing MTT assay on HepG2, i've kept control ( cell + Medium) , Negative control (Without cells+ Medium), Positive control (BAP), Vehicle control (Methanol, and DMSO). can anybody tell me exact procedure to calculate toxicity or Cell viability after taking O.D. at 540nm and 670nm( for back ground correction)

    Parag Kolekar

    Thank you. I will send soon.

  • Daniil R. Petrenyov added an answer:
    3
    Any advice on conflicting results between Alamar blue and hoechst DNA?

    Hello, 

    I've incubated MG63 with increasing concentrations of HA nanoparticles. After 24 hours I conducted an alamar blue assay and subsequently a Hoechst DNA assay.
    After analysing the results, the alamar blue shows a decreasing trend in metabolic activity with increasing NP concentration. However after looking at the hoechst DNA assay I see an opposing trend with DNA content and hence relative cell number actually increasing with increasing NP concentration. 

    Could someone possibly shed light as to what is happening? Could it possibly be interference with NPs?

    Thanks  

    Daniil R. Petrenyov

    I agree with Adeel Masood Butt.

    Correct controls could shed more light on these findings.

    Some additional hypothesis to test:

    1. NPs may affect mitochondria efficiency and so decrease total metabolic activity.

    2. Alamar blue could contains some small molecule electron carriers to enhance reduction of Rezazurin,so NPs could affects active concentration and/or trafficking of these carriers.

    3. NPs could account to false positive signal during measuring of DNA content or increase quantum efficiency of hoechst:DNA complex.

    I guess some of these ideas could be tested with mitochondrial potential dyes and some  in cell free system.

    Hope it would be helpful.

  • Andrei Alexandru CONSTANTINESCU added an answer:
    2
    How can I calculate plating efficiency for floating cells such as those from leukemia?

    I am doing a viability study using leukemia cells and planning on plating them into a 12 well plate.

    1. I was wondering how plating efficiency can be calculated for floating cells such as leukemia cells that do not adhere and form colonies?

    I found an online calculation that shows

    Plating efficiency= Cell Count on D1/ Cell Count on D0*100 

    Can this be used for determining the plating efficiency for floating cells?

    2. Is there a different method used for floating cells during in vitro viability studies that would be equivalent to plating efficiency? 

    3. My main concern is to be able to factor for that 12 well plate might have different efficiency than a 6 well plate or T25 flask. I want to be able to effectively factor in the efficiency of the 12 well plate into my viability calculations and remove any inconsistencies caused by the plate of choice. What would be a good method to ensure that?  

    OR Is this not a problem with floating cells as much, as it is for adhering cells which needed a specific surface area and seeding density?

    Andrei Alexandru CONSTANTINESCU

    When I use suspension cell culturing I consider the number of cells per mL. Usually I seed 200.000-400.000 per mL (depending on culturing period, stimulation length, stimulation molecule concentration etc.), and treat at ~800.000 cells/mL. Higher seeding cell concentration might lead to higher doubling rate, reaching the stationary growth phase earlier. Higher cell concentration at stimulation point might alter the cell response. Critical is to use the same treatment conditions (cell no per well per volume, treatment length etc.) in every independent manipulation for the respective assay. Important is to have you cell culture in the Log growth phase. Is up to you to set the correct parameters.

  • P. Bagavandoss added an answer:
    4
    What is the meaning of negative value percentage of inhibition? The value of absorbance of sample are higher than the absorbance of negative control?

    I have carried out the MTS test several times. The same dilution factor and sample. The results show that the absorbance of sample are higher than the control. When I plotted the graph percentage of inhibition vs concentration the value sometimes negative. Is it shows that the "negative value e.g. -10%" is growth of cell? Attached herewith an example of the graph I get.

    P. Bagavandoss

    There is a concept called hormetic response, an example of biphasic response. In this view, a given chemical can stimulate at lower concentrations but inhibit at higher concentrations. This is a true response. This is more of a norm than exception.

  • Tobias Pusterla added an answer:
    3
    Can I combine two luminescent cell-based assays?

    I am currently using an Anthos Lucy 2 Microplate Luminometer to assess cytotoxicity in Organ of Corti-2 cells (inner ear).  I would like to study cell viability and cell death amongst these cells when exposed to different stressors at different concentrations.  However, this particular microplate reader is only compatible with luminometric (Dual Luciferase) and colourmetric (405nm-690nm) assays.

    Therefore, I was wondering whether anyone knows if the CellTiter-Glo Luminescent Cell Viability Assay (which measures ATP¨) can be multiplexed with the Caspase-Glo 3/7 Assay (which measures caspase-3 activity)?  They both act via bioluminescence.  I know that combining two or more luminescent-based assays can be difficult.

    Many thanks for your help!

    Tobias Pusterla

    Hi,

    I attach an example of a combined cell viability and toxicity measurements (luminescence and fluorescence). I do not think either that a multiplexing with lum and abs would be possible for your assays.To answer your question, you have to check if the two luminescent lights emitted have different emission wavelengths and if those wavelength are far away enough to be resolved by a luminometer. If one emission is "leaking" into the other (the two emission peaks are too close) you cannot resolve the two lum peaks. This will result in higher background noise and less accurate measurements. If the two wavelength are resolvable, you could use a microplate reader that allows the use of luminescence filters, by this you´ll be able to separate the two wavelengths during two sequential measurements. If your luminometer does not allow the use of filters for luminescence, then I am afraid you cannot di it.

    Hope this helps!

  • Closed account added an answer:
    2
    How to handle fluorescence of nano fibrillated cellulose (NFC) scaffolds during Live/Dead staining?

    To evaluate cell viability in a 3D printed NFC scaffold, I want to use a live/dead assay to stain the cells (dyes: calcein and Ethidium homodimer). Unfortunately I get a background staining from the NFC scaffold (presumably due to DNA residues of the NFC). Therefore, I was wondering if anyone knows how to handle that issue or knows a different approach for a selective live/dead staining? (I do not want to break down the scaffold)

    Deleted
  • Mya Fekry added an answer:
    6
    What is a good alternative for MTT assay to determine cell viability?

    Currently we are using the MTT assay to determine cell viability, but we experience the loss of the formed formazan and we are looking for an alternative assay to replace our MTT.

    Mya Fekry

    Hi Anke, I have used the CCK-8 assay and can recommend this also. It has better sensitivity than some of the other WST salt assays. Alternatively, other options like Trypan blue staining or Alamar blue may work just as well for you.

  • Mohamed Maarof added an answer:
    4
    Can you help me to calculate the CCI 50 in MTT?

    I've read my MTT assay on 450 nm and 620 nm as reference.

    Can you help me how to calculate the CCI 50 for both the virus and the extract?

    The reading on 620 was much higher than on 450.
    Would you please me for getting the best results?
    P.S. I don't have any available wavelength in my lab only 405, 450, 620 nm

    Mohamed Maarof

    Actually I don't have ay software for calculation .
    can you kindly suggest me one to be freely downloaded ?
    and how can I use it to get my concentration ?

  • Gerald Doh added an answer:
    5
    Any advice on the UV to inflict DNA damage to HEK293 cells in culture?

    I am studying a protein and from imaging I can see that my protein is recruited to sites of DNA damage. I wish to UV irradiate HEK293 cells in culture prior to collection and analyses by RNA-seq and mass spectrometer. Does anyone have an idea of how to (protocol and instrumentation) UV irradiate cells in culture for such studies? 

    Gerald Doh

    Thanks guys. I am trying the protocol today. Once more, thanks

  • Zeravan Mohammed added an answer:
    9
    How can I stimulate endothelial cells by LPS?

    I want to stimulate a Human endothelial cell line (HUVEC) by LPS and test my drugs of interest to quantify the anti-inflammatory effect, I used the DMEM medium and 10% FBS for HUVECs, at first I test the viability of cells by MTT assay, for this I stimulated with 1µg/ml, 5 µg/ml and 10 µg/ml of LPS. I just use the 5 µg/ml and 10 µg/ml concentration to see if LPS has some effect on endothelial cells but there was no difference with the control group. Where is the problem? I bought the LPS form Sigma. Does the HUVEC stimulation need a special protocol?

    Zeravan Mohammed

    can anyone tell me how LPS act on endothelial cells as we know endothelial cells lack CD14 (mCD14)???

  • Nesrine Abboud added an answer:
    3
    Any idea about cellular viability markers, which are not influenced by the mitochondrial activity and by the dynamics of the cellular membrane?

    Usually I use the calcein or the MTT, but my present experiments consist in determining the quantity of mitochondrial ROS  in my stressed cells and my results of cellular viability seem not logical. I 'm looking for an other marker of cellular viability. Thanks in advance for your help

    Nesrine Abboud

    Thank you very much for your answers

  • Antonio Bovo added an answer:
    5
    Does anyone have a good protocol to study the viability of cells encapsulated in a hydrogel?

    I have to know the viability of cells encapsulated in a hydrogel.

    I've tried to study the viability of cells encapsulated in a hydrogel with alamar blue but I think that resazurin doesn't penetrate in the hydrogel and hydrogel interferes with the fluorescence of resorufin.

    Antonio Bovo

    Thank you all for the answers!

    Can you suggest me the best way to use the live/dead assay to be sure that it penetrate into the hydrogel and reach cells  encapsulated in the hydrogel?

  • Patrick Heinrich added an answer:
    13
    Anyone familiar with LDH assay and MTT assay?

    Hello

    I run both cell viability using MTT and membrane integrity using LDH analysis, but I find the same compound shows higher levels of LDH release compared to the cell viability data. Can anyone explain this for me please?

    Patrick Heinrich

    Hi,

    as you described, you calculate your effects with your negative control signals set to 100 % viability, however, you do not assess viability in LDH as you would in the MTT assay, but you measure cytotoxicity or cell mortality, respectively. In LDH, the actual negative control signals are not (!) what you refer to, but the positive control signals, which represent 100 % mortality by definition. Actually, you would have to set your negative control signals to 0 % cytotoxicity for correct calculation, so they technically respresent something similar to a blank measurement (activity present in medium with fully viable cells).

    Again, your positive control signal cannot ever become 500 %, because it is set to 100 % mortality by definition. Right now, you are making a mistake by setting your negative control signals to 100 %, which is right for most assays (e.g. MTT), but not for LDH.

    Refer to the figure posted above and compare the two test methods LDH and almar Blue. As the signal increases for LDH with increasing treatment concentrations, almar Blue signals decrease almost symmetrically. From what you explain, it sounds to me that you observe something similar in those two tests (just swap almar Blue for your MTT assay here), which is however something you would actually expect given from the different nature of both approaches.

    To make things more clear, try to plot both results into the same graph similar to the graph on the website (or individual graphs) and post them here, it is always easier to discuss such matters on actual data.

  • Tarek A Elkhooly added an answer:
    4
    How to check cell viability assay without killing the cells?

    I want to check the cell viability of 3T3 cells without killing them or deteriorating their proliferation. I tried CCK-8 kit which keeps the cell alive but it affects the cell growth and proliferation. Please suggest some assay where I can use it without killing cells. 

    Tarek A Elkhooly

    you can try CCK-8 assay,please read the attached file

  • Saswata Chatterjee added an answer:
    4
    In a time-dependent MTT assay, after each time interval change media and keep it for 24 h incubation then add MTT or add MTT just after each interval?

    Which one is correct?

    Cell seed-24 hr incubation-Drug treatment-after 3H media changed and kept back in incubator upto 24h-after 6H media changed and kept back in incubator upto 24h-- after 24h both plates taken out media changed and MTT added--4h incubation-- media discard--DMSO added and mixed-- reading at 550 nm

    Cell seed---24 hr incubation---Drug treatment---after 3H media changed and MTT added (4hr incubation and DMSO added)--- after 6H media changed and MTT added (4hr incubation and DMSO added)-- reading at 550 nm

    Saswata Chatterjee

    thank you everyone for your valuable comments

  • Amit Kumar added an answer:
    6
    Upto what passage number do BNL 1ME A.7R.1 cells show normal behavior and normal characteristics to be used for cell viability assay?

    I am trying to run MTT assays on a passage number 70 for the cell line and I do not see the same results as I used to see at a lower passage number for the same cell line? some insights would be helpful. Thanks

  • Cássio Prinholato da Silva added an answer:
    3
    Anyone expert in Penicillium chrysogenum, how can I count total cells and measure viability?

    Anyone expert in Penicillium chrysogenum, how can I count total cells and measure viability? Thanks

    Cássio Prinholato da Silva

    All the best for you!

  • Shivani Pandya added an answer:
    4
    Polymer coated Fe nanoparticles are insoluble in various organic solvents. How can I prepare these samples for cell viability assay?

    Fe nanoparticles coated with polymer have been developed as drug carriers. Particle size <50 nm.

    They are highly insoluble in organic solvents including DMSO or Ethanol.

    Due to their solubility issue, their dilution is also a hassle.

    We have tried sonicating the samples prior to dosing the cells.

    Everytime the result is "nontoxic" even with dose as high as 2mg/ml.

    I doubt that the nanoparticles are not interacting with the cells to manifest biological effects.

    Shivani Pandya

    On the bases of my work experience with Iron (II, III) oxide nanoparticles, they are highly insoluble in water and most of the organic solvents but they might be disperse in DMSO (aprotic polar solvent) and for that, may my paper will provide some help to you. Go through the given link. (http://www.sciencedirect.com/science/article/pii/S0167732215302038).

    Second thing as you said you have coated your MNPs with Polymer, so please go through the properties of polymer u have used because polymers are of different types some are water soluble and some are fat soluble so please check which type it is.

    Third one is, as you have taken 2 mg/ml MNPs dosage for cell uptake study and it was gave you "Non-toxicity" because these particles are biocompatible in nature and they were approved by FDA for use in bio-medical applications. So there are very less chance to be a toxic for cell unless you have not coat your NPs with such toxic molecule. however, when we are talking about nanoparticles they are highly reactive, so they probably interact with cell and cellular material, but in this case they are not causing cell damage and also not altering cell cycle and so they are nontoxic.

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