Differentiation - Science topic

Hi Drake,

I have the same questions, and i also could not find a fully reviewed paper about it, have you figure it out yet?

I tried to ask ChatGPT, and here is the answer, hope is useful for both of us:

Dimethyl sulfoxide (DMSO) is a solvent commonly used in cell biology experiments, and it can also induce the differentiation of certain cell types. For example, in the human acute myeloid leukemia cell line HL60, DMSO can induce these cells to differentiate into neutrophil-like cells.

The mechanisms by which DMSO induces differentiation primarily include the following:

Through these mechanisms, DMSO induces morphological and functional changes in HL60 cells, leading them to exhibit characteristics of neutrophils. These characteristics include morphological changes such as cell shape and nuclear maturation, as well as the upregulation of specific neutrophil markers.

Dear Alexandr,

Thank you for the answer. Unfortunately, I already know these works really well!

I can't use neural crest markers in IF as most of them are either cytoplasmic or downregulated before the enteric neurons become terminally differentiated.

Sox10 works quite well up to a certain point, but it's absent in mature neurons: its expression is maintained in enteric glia only.

Danuta Sastre

Great, please let me know if you have any further questions.

No, they are not

Basal calcium is usually low in the cytoplasm of unstimulated cells, most of it is stored in the ER and in the extracellular environment. A good way to assess the differentiation of iPSCs to neurons is by testing their ability to respond to external stimuli or to fire spontaneously after synapse formation in culture. These activities would determine calcium transients that can be measured and quantified - e.g. comparing the response to the baseline (stimulus vs absence of stimulus). You can check this paper about an iPSCs-derived disease model.

Think about it in the other way, If it is not continuous at a given point can we find the derivative of the function ( instant change) in that point?

The growth factor information here is a starting place.https://www.biorxiv.org/content/10.1101/699173v1.full

for epithelialization, retinoic acid or thrombin are important: https://www.frontiersin.org/articles/10.3389/fncel.2018.00045/full

Anwar A. El-Hamaky

Hi Sanghyeon Oh,

I am using the same protocol and have the same problem. Those defective patches start around Day8 -Day10 and until Day14 I lose almost all of them. Once, I tried to collect them on Day 12. It kind of worked, but unfortunately not all of them can stay even until Day 12. I just wanted to ask if you could solve your problem?

Thank you.

Has your problem been solved? I have the same problem as you. But my differentiation system: DMEM, 0.5% FBS (Heat-inactivated), 1x antibiotics。

I am also having the same problem. Its great honor if you could share with me.

Dear Prof. Bruno Cessac, in addition to all the interesting answers in this thread, there is a paper that explains historically the evolution of the Ehrenfest classification of the phase transitions, it might be good to add it since it talks about the Pippard extension of the classification when there are singular points in the specific heat at the T_c as in the ferromagnetic/antiferromagnetic-to-paramagnetic transitions in Ni (ferrom.), MnO (antiferrom.) and other crystals.

The answer to a question about the numerical algorithms for resolving the issue of differentiability of a function is typically provided by the textbooks on experimental mathematics.

I recommend in particular: Chapter 5: “Exploring Strange Functions on the Computer” in the book: “Experimental Mathematic in Action”.

For the review please see

You can also get a copy of the text in a form of a preprint from

Judging by the quote placed in the beginning of Chapter 5, the issue of investigation of the “strange functions” was equally challenging i 1850s as it is 170 years later:

“It appears to me that the Metaphysics of Weierstrass’s function

still hides many riddles and I cannot help thinking that enter-

ing deeper into the matter will finally lead us to a limit of our

intellect, similar to the bound drawn by the concepts of force

and matter in Mechanics. These functions seem to me, to say

it briefly, to impose separations, not, like the rational numbers”

(Paul du Bois-Reymond, [129], 1875)

The situation described in your question is even more complicated because the function is represented only by a few values on a rectangular grid and it is additionally assumed that the function is not differentiable at a certain point. In this situation I can suggest to use the techniques employed in the theory of generalized functions (distributions).

For a very practical example you can consult a blog: “How to differentiate a non-differentiable function”:

In order to answer your question completely I would like to know what is the equation, boundary conditions and the numerical scheme used to obtain a set of the grid point values mentioned in the question.

it is a good idea to ask, as trypsin is not suitable for this task. Use papain, for example see our earlier paper PMID: 19609935, DOI: 10.1002/stem.177 . Trituration ("mechanically pipetting slowly") should not be used, this is an old approach, causing a higg## of cells to die. N.spheres have more and less differentiated cells, more differentiated cells will die (high number). Others use accutase, but papain (with or without very gentle trituration) is #1 choice. You can also have the n.spheres attach and release neural cells (if the goal is to culture) or embed in OCT and generate cryosections and do IHC (if the goal is counting % of different cell types). If you are doing flow - definitely do not do trypsin, as it digests and destroys many key epitopes on the surface of neural cells (see Maric and Barker papers for this). Good luck

As you said, a professor is expected to provide equitable professional service to his/her students. To this end, he/she has to know the entry behaviors of the students under his guardianship. Once he/she knows the students, he/she has to do a detailed plan that provides varied opportunities for different learners. That is, the professor should varied contents, activities, assessments, and what you name. Then, he/she is expected to conduct lessons using the plan as a guide. That is what it means by differentiated instruction.

The best program is Image-J software.

Hey Elmira,

Could you post the picture of how cells look.

I had some issues with my RPE1 cells and a person suggested it might be some type of contamination but now I am not sure.

Thank You!

Thanks Wolfgang Doerr

Do you know if the transit/differentiating cell is morphologically different from the new stem cell?

Also, is there a immotalized cell line that also exhibit such ACD?

The only way I see - as in almost all tissues apart from bone marrow there is no specific stem cell marker - the only way is to establish a cell culture procedure and look for clonogenic, i.e colony-forming cells. They may not reflect the true number of stem cells, but will allow for the estimation of relative changes.

W.

There are lot papers which described about cell cycle activity during differentiation. You are right that cell proliferation need to be stop, so cell can go for differentiation.

How any differentiated cells expresses a particular markers which undifferentiated cells do not expresses. Similarly, for proliferation and apoptosis phenomenon. Apoptosis assays will determine whether differentiation induction caused any death or not.

While, to track the cell proliferation, you can use FLOW CYTOMETRY analysis for Ki67, which is prominent proliferation marker, also PCNA & MCM2 expression. Even several dyes are available such as BrdU, EdU can be used.

Ki67 expression before and after differentiation will give very good answer whether cell exit the cell cycle or not...

Yes by sorting the cells.

* A virtual displacement is any field of displacements, which satisfy the boundary conditions, but is imaginary (do not actually occur). Moreover, a virtual displacement means an instantaneous (time is held constant) change in coordinates so the state of stress does not change (a real displacement would change the state of stress).

Then, the work of all (real) forces on virtual displacements should be zero if the system is in a state of equilibrium.

* The variation in displacement is an infinitesimal change in the displacement field so the change in the state of stress can be neglected. Then, the variation in displacement can be interpreted as virtual dispalcement.

Here are the notes

this can be helpful.

Shen-An is correct in that you should get rid of 2-Me, it serves no purpose. Also RPMI is already buffered so HEPES likely wont do much.

We tend never to actually replace the media in the first few days, but just top up existing media, there is probably something made by the macrophages themselves that helps with survival (maybe GM-CSF or more M-CSF, or the macrophages like eating the dead neutrophils, its unclear).

We usually seed 30 million bone marrow cells in 30ml media in a T175 on day 0 (20ng/ml MCSF) so this equates to 4.2 million in a T25 in 4.2ml (pretty much what Shen-An uses). Feeding 10ml on day 3 and 6 works the best and by day 7 you will have lots of macrophages to treat with whatever you want. There will be 50% cell death by day 3 (all the neutrophils and other precursors will die - this is normal and the straight line of dead cells you are seeing, I hypothesise that the new macrophages start eating these dead cells), but if you give lots of media on the day 3 feed they will start to proliferate. On day 7 you will have 15 million macrophages. To much M-CSF feeding triggers more proliferation but may leave the cells slightly 'exhausted'. You may have to experiment with concentrations based on the M-CSF batch also.

M-CSF is a vital growth and survival factor (known for >25 years), its actually required to maintain mitochondrial function. Taking M-CSF away will immediately reduce mitochondrial function and eventually trigger apoptosis (unless cells are so dense they make their own M-CSF, also its possible LPS stimulates autocrine growth factor signalling). IL-4 also acts as a 'growth factor' and can work independently of M-CSF.

If you rest the macrophages with no LPS, no IL-4 and no M-CSF they will begin to die, I am not surprised at all you see this. Everyone can tell you if you go away on the weekend and forget to feed your macrophages M-CSF they will die. Maybe by 16 hours you wont see too much evidence of dead cells, but push that to 36 hours and every cell will be dead. It's possible in Shen-An's culture conditions have sufficient autocrine signalling to allow survival, lab variation with this is common and things get really crazy when people use L929 media.

But really there is no reason to remove M-CSF. I cannot think of a time in the body of a human or mouse when you have a infection and all the M-CSF (or IL-34) that is present in the tissue (at pretty decent concentrations - a steady 10ng/ml in the peritoneum) magically disappears to allow a 'pure' LPS or IL-4 signal. These things work in concert, keep it in, keep the cells happy. If your results are different with M-CSF then its possible this is more physiologically relevant, and I would be happy to have a discussion with anyone who disagrees. (This also brings up the possibility of combining GM-CSF and M-CSF, but that's another issue)

Hope this helps,

Best of Luck,

Luke

I would look at Mmp9 and Mmp12 after losing stimulation. Also consider Mcl1.

To the best of my knowledge, so far people haven't fully understood the lineage tracing and fate mapping of the bone marrow stromal cells that are generally considered as of mesenchymal origin (i.e. from the mesoderm), not to mention to even specifically control their differentiation into some subsets such as osteoblasts. BMSCs are differentiated from the hematopoietic by using triple negative surface markers CD45-, CD31-, and Ter119-, but this most recent paper: https://www.cell.com/immunity/fulltext/S1074-7613(18)30382-0

tells us the majority of these BMSCs are from hematopoietic origin! BMSCs that provide key niche factors including CXCL12, SCF, and others only count for 20% of these triple-negative populations.

Hope this provides at least some insight to your question.

I should have been more clear. PMA differentiates the monocytes into macrophages. Activation (to be stimulated) usually requires some thing, like LPS or any other stimulants. PMA treated macrophages do produce some cytokines, but you can push for more with stimulation. I usually don't consider PMA treated macrophages as "activated", but that has to do with the model I was working in. However, using a combination of IFNg and IL-4 and others, depending on the cocktail, you can shift the macrophages into a M1 or M2.

Normalized differential pond index (NDPI) is also used to estimate the area under water bodies. It is calculated using the green and shortwave infrared (SWIR) bands of satellite imagery .

Test to be applied depends upon the nature of hypothesis to be tested, type of data used, any prior information known , the parent population of the sampled data etc.

As I know, this is a case of t test.

Dear Friends,

I have published a paper recently.

it shows the attitude of stem cells researchers.

All the best!

Mahdi.

Differentiation implies the coordinated motion of 'genome as a whole' and cannot be restricted to the identification of single 'mastr genes' this is why I am convinced the OMICS can give a very relevant contribution to the problem if and only if we acquire a statistical mechanics global approach to the quantification of correlation and order during the differentiaion process, here are some worked examples:

I use 0.07 mM CaCl2 for my HEKa and neonatal foreskin HEK cells as the standard growth concentrations and then if I do decide to differentiate them I will incubate them in 0.03 mM Ca for 24 hours and then switch to the high calcium concentration of 1.2 mM, which from what I've seen is standard. I always buy medium without Ca and then add as much CaCl2 as I need for each condition. I also use serum free media. Hope this helps.

Hy,

I worked with temperature sensitive immortalised neuronal progenitor cells from the rat midbrain (CSM14.1-cells).

They proliferated also at 37°C (not only at 33°C) , but really only differentiated into a neuronal fate at 39°C when the serum was reduced from 10% to 1%. When we cultured them with 10% serum they continued to proliferate.

So, you have also to consider the serum content of your media and not only the temperature.

Sincerely Yours/Beste Grüße

Stefan (with an f)

Hi,

@Meaghan Roy-O'Reilly,

I had performed BCA and loaded equal amount of protein from both the samples.

I have read somewhere that actin levels, do vary on differentiation.

So i had thought that total protein levels for that phospho protein, will serve as loading controls, as used by others in similar study, but in my case I am getting even total protein levels varying between the samples.

he EEG signal itself has several components separated by frequency. Delta waves are characteristic of deep sleep and are high amplitude waves in the frequency range 0≤f≤4 Hz. Theta waves occur within the 4-8 Hz frequency band during meditation, idling, or drowsiness. Alpha waves have frequency range 8-14 Hz and take place while relaxing or reflecting. Another way to boost alpha waves is to close the eyes. Beta waves reside in the 13-30 Hz frequency band and are characteristic of the user being alert or active. They become present while the user is concentrating. Gamma waves in the 30-100 Hz range occur during sensory processing of sound and sight. Lastly, mu waves occur in the 8-13 Hz frequency range while motor neurons are at rest.he EEG signal itself has several components separated by frequency. Delta waves are characteristic of deep sleep and are high amplitude waves in the frequency range 0≤f≤4 Hz. Theta waves occur within the 4-8 Hz frequency band during meditation, idling, or drowsiness. Alpha waves have frequency range 8-14 Hz and take place while relaxing or reflecting. Another way to boost alpha waves is to close the eyes. Beta waves reside in the 13-30 Hz frequency band and are characteristic of the user being alert or active. They become present while the user is concentrating. Gamma waves in the 30-100 Hz range occur during sensory processing of sound and sight. Lastly, mu waves occur in the 8-13 Hz frequency range while motor neurons are at rest.Hope it may be possible

Hello

See these links 

Good luck

I am agree with Areeful but  I found very few disruption of cells in my experiment, which is negligible. I am using Remi 8r centrifuge machine. Therefore, you should try to centrifuge at low rpm for more time to get cell pellet, that will decrease the disruption of cells.

Yes its possible. Unfortunately there is the possibility that one of the barcode was attributed to Aspergillus but comes from a different species, basically a mistake by th eperson that entered the sequence. Try the the Bar Code of life it is well curated: http://www.boldsystems.org/index.php/IDS_OpenIdEngine

The second possibility is that the two aspergillus species cannot be separated at the species level using their  bar code, however 100% is surprising, I would go for the above mentionned option.

This is an example of a linear system of ODEs. If we have  x'=Mx, where M is a matrix of coefficients and x is a vector of unknowns, then the substitution of x=Xe^{lambda*t), gives      (M-lambda*I)X=0, from which we deduce that the condition for nonzero solutions for the constant vector, X, corresponds to solving det(M-lambda*I)=0. Thus the lambda values are the eigenvalues, the computed values of X are the eigenvectors, and (M-lambda*I) is the characteristic matrix.

In the problem which you are solving you have second derivatives and this is why lambda^2 appears, rather than lambda in my example. This author has also substituted e^{lambda*t) into (3.8) and written the consequence of that substitution in a matrix/vector form which is analogous to my (M-lambda*I)X=0.

Dear Dana, many thanks, I will check them now. 

all the best

Imran

what was a percentage of them?

Hi Arbia,

I might be missing something here because I’m not sure if I understand in part of what you wrote in your posts. Could you clarify if you took the following approach?

Technically, this is a PDE, where the Maxwell-Faraday’s law (in point form) is given by

curl(E) = − ∂B/∂t

∇ × E = − ∂B/∂t

[(1/r)*(∂E_z/∂θ) − (∂E_θ/∂z)] r + [(∂E_r/∂z) − (∂E_z/∂r)] θ + (1/r)*[(∂(r*E_θ)/∂r) − (∂E_r/∂θ)] z = − ∂B/∂t

Let's say that the magnetic field in a certain region is given by the sinusoidal expression

B = B₀ cos(kr − ωt) z.

Equating the z component, we have

(1/r)*[(∂(r*E_θ)/∂r) − (∂E_r/∂θ)] z = − ∂[B₀ cos(kr − ωt) z]/∂t

(1/r)*[∂(r*E_θ)/∂r] − (1/r)*(∂E_r/∂θ) = − ω*B₀ sin(kr − ωt)

For some unspecified assumptions, the PDE is transformed to a first-order linear ODE.

(1/r)*[d(r*E_θ)/dr] = (1/r)*E_θ − ω*B₀ sin(kr − ωt)

The general solution is given by

E_θ = (ω/k)*B₀ cos(kr − ωt) + constant.

Caro Paolo,

Thank you for rapid answer, I am very happy with your answers.

Best regards,

Antonio Ronaldo.

You can probably test the use o gibberellins to artificially bypass vernalisation

The nonlinear Gao beam is an interesting application in beam theory that allows for the possibility of buckling of the beam under a load. While I have seen this as a PDE, you can certainly consider it at steady-state.

Hi Yash,

It would really help the reader to understand better, if you could describe the problem exactly what it is. Based on the above description, naturally, people would assume it is a coupled system of two second-order differential equations. If they are homogeneous linear ordinary differential equations and the initial values are given, then one way to solve them is to use Laplace transforms.

The following example shows how to solve the Roll-Yaw motion at equilibrium condition, which is a system of two linearly coupled ordinary differential equations, where ϕ is the roll angle displacement, ψ is the yaw angle displacement, ω is the natural angular frequency of the Roll-Yaw system, A is associated with the damping characteristics, and B & C are associated with the stiffness characteristics.

Dear Joachim,

Thank you for your comments regarding the domain for the  commutator D^{alpha}J^{alpha}=J^{alpha}D^{alpha} with alpha being fractional.  

Since the domain contains the set of polynomials, it may extend to the set of analytic functions along with appropriate closure, which will depend on how to compute the commutator [D^{alpha}, J^{alpha}] in certain integral form.

The review article "A Review of Definitions for Fractional Derivatives and Integral" also gives detailed exposition about the calculus-wise origin for the definition of the fractional derivatives.

Best Regards, Shijun 

It isn't possible. The Navier-Stokes equations are a set of coupled, nonlinear partial differential equations. Your quoted equations are uncoupled, linear ordinary differential equations. It may be that your system has been derived from the NS equations, but this would have been subject to all sorts of simplifying assumptions for a special type of flow. Therefore, at best, your equations form a very small subset of the NS equations, and the latter cannot be reconstructed from the former.

 http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_Oil_Red_O_Stain_for_In_Vitro_Adipogenesis.pdf

follow this one

That sounds strange! Don't you search for solutions of the two differential equations? Roots is a concept for solutions of algebraic type of equations.

One may say that there is only one equation, since the first reduces to the second when k=0. The expression given by Akhlaq is not a correct solution. In the special case that (bg-ak)=0 there is a simple solution,

In the case that k=0 one may express x as function of y, but that expression involves a special function (the exponential integral). For k!=0 one may still express x as an integral involving y, but that integral cannot be expressed in terms of any named function.

From a practical point of view a numerical solution may be the best option. However, for qualitative understanding one can think of x as the time coordinate. Then the first equation can simply be interpreted as Newton's second law for a particle with position y, moving in a potential

By plotting this potential for some relevant values of the parameters, and taking into account the initial conditions, you can learn a lot about the qualitative behaviour of the solution. Which can be very useful in combination with numerical integration.

Dear Sarah,

I think that you have to impose some extra conditions on f, k and even to the notion of the fractional derivative to obtain the corresponding fractional differential equation (FDE). Even in the simplest cases it is not clear what is the corresponding FDE. Let us consider a couple of examples.

First, take k(x,t)=1 and f is differentiable. Then differentiating

(1) D^\alpha y(x)=f(x)+\int_0^x k(x,t)y(t)dt

yields

(2) DD^\alpha y(x)=f'(x)+y(x).

But since the fractional integration and differentiation does not commute in general, DD^\alpha is not necessarily a fractional derivative of order \alpha+1. If D^\alpha denotes the Riemann-Liouville fractional derivative, then (2) corresponds to FDE:

(3) D^(\alpha+1) y(x)-y(x)=f'(x).

However, if D^\alpha denotes e.g. the Caputo fractional derivative, then DD^\alpha is not necessarily D^(1+\alpha).

Second, take k(x,t)=c(x-t)^(\beta-1) for some constant c. If c=1/\Gamma(\beta), then the integral term in (1) corresponds to the Riemann-Liouville fractional integral of order \beta, which is denoted as I^\beta y. If the fractional derivative D^\beta f exists and D^\beta is the left inverse of I^\beta, then (1) converts into

(4) D^\beta D^\alpha y(x)=D^\beta f(x)+y(x).

But for the same reason as in the first case, D^\beta D^\alpha is not D^(\alpha+\beta) in general.

Hopefully you will find this useful in your further considerations.

Best regards, Jukka

I am currently following the protocol from Kriks et al 2011, where they have used small molecules like LDN, SB, SHH, PUR, FGF8 (in KnockOut medium) among others to differentiate iPSCs into dopaminergic neurons. 

So, I was wondering what would one expect if these molecules are not added to the medium?

Hi Athul,

the best marker to study muscle differentiation and then myotube formation is Myosin Heavy Chain (MF20 antibody clone) works perfectly in IF and WB, beside you can use also Desmin is commonly accepted also if personaly I do not like so mutch. Furthermore there are trascription factor (MRF) likewise MyoD and Myogenin that works quite well in IF and WB.

Good luck

Thank you for the reply

I don't see anything wrong with student access to R^G, given the general access rules which seem to be applied. What is wrong is to use this access to (try to) make other members explicitly solve ones homework problems. It this case I did provide a solution, because I thought it might demonstrate a different (physicists) approach, complementary to the general formulas of calculus - which the questioner should have learned in some class.

Alternatives are to respond with a warning to the questioner about the dangers of cheating, and/or to flag the question(er) to R^G. Students must learn to use new technology, but they should not forget appropriate behaviour and ethical issues.

Hi ! If we denote  f(t)=\int_a^x g(\tau) d\tau , where g(x) is a non negative  continuously differentiable  on (a.b) function with horizontal   asymptote  in a and vertical asymptote in b  then the first four conditions for f(x) are satisfied and for K > 0 the last inequality becomes a Riccati type inequality g'(c) + g^2(x)/K >=0 with respect to g(x). One of solutions of this inequality is g(x) = \lambda - \tan(\mu x - a), where \lambda > 0 and \mu > 0 are some constants which we can find from the conditions of problem .

How to find a solution (if such solution exists) of a differential inequality ? - ResearchGate. Available from: https://www.researchgate.net/post/How_to_find_a_solution_if_such_solution_exists_of_a_differential_inequality [accessed Nov 15, 2016].

Hi Vindya, in general when you are plating cells in 10%FBS, they will adhere to serum vitronectin, which is  an RGD containing serum protein and is very effective at coating TC plastic.

There might be differences in cell  morphology whether you coat cells along with the  FBS-media or put the cells in after FBS-media is already on the plates.

May I suggest that you try antibodies to AE01/AE03 which are very useful cytokeratin markers for the detection of adenocarcinomas.

@H.Mohammad: Thanks

Claudia, 

Since the analyzed signal is always finite, I am almost sure that the entropy of the signal will depend on the sampling rate. The answer will differ from sampling rate to another, and the higher the sampling rate and the more accurate result you may get if the signal length in time is sufficient. Anyways, your sampling frequency should not be below the Nyquist frequency. 

Good Luck!

I should point out that for maximum principle, C might be a convex compact subset in a locally convex space, or an unbounded closed  "generalization " of such a subset. Going back to the minimum point of a strictly convex and differentiable function f on the open (convex) subset C, then f '(x) = 0 if and only if x is a strictly global minimum point of f. In this particular case, such a minimum point is unique.

The following references may be relevant

http://dx.doi.org/10.1016/j.cnsns.2010.01.037  (http://arxiv.org/abs/0911.1848)

http://dx.doi.org/10.1088/0031-8949/89/03/038003   (http://arxiv.org/abs/1308.5126)

For the beam equation, see, e.g.,

Dear,

The differential or linear attacks are performed by world-class crypto researchers, and the competition is rather rough. So, people do not take the time, or are not always willing to share their software with everybody.

There is an FPGA Implementation of the Linear Cryptanalysis in this link :

Consider also using a stepwise logistic regression in which you use 0=group 1 and 1=group 2, with twenty independent variables. If there is multicollinearity among the independent variables, it may be better to remove some variables before running the logistic regression. The odds ratio will tell you which variables are most important. 

Assuming that you are changing media every day, are you also removing the differentiated colonies as you notice them? If they aren't removed, they will influence surrounding cells and colonies to differentiate. Also, after passage they may grow faster than the pluripotent cells and take over the culture. We use a dissecting scope in a laminar flow hood to completely remove these colonies several times a week. Also, if you used episomal vectors to reprogram, the differentiation may be a result of the vectors being kicked out of the cells (as they should)...if that cell line is not correctly reprogrammed it will lose it's pluripotency that was previously maintained by expression from the vectors.

You may need to check 1. Passage number of your cells; better take passage no. 2-10. 2. Check your media composition and culturing conditions. 3. Initial support with 15%FBS, instead of 10% may help to resolve your problem. Best Wishes

Yes, you can follow the same protocol.

Please note that conversion of human fibroblasts into induced neurons, referred to in the publication (link) as iN cells.

Rafik

morphological you can differentiate by observing  the thin smear stained with Romanowsky stain under the microscope also the clinical symptom is very important . Read a book known as Helminths, Arthropods and protozoa of Domesticated Animals (Monnig) 6th Edition E.J.L.Soulsby

Maybe you should make two simple experiments. You can asses proliferation via MTT assay or BrdU staining.

The differentiation can be induce using an adipogenic differentiation protocol using insulin, dexamethasone and IBMX and followed by a maintenance medium with insulin.

After differentiation, cells can be fixed in PFA and stained with oil red o (ORO) and the percentage of differentiated cells can be achieved evaluating the ration between cells ORO positive and total nuclei.

Hi Nai-Kei Wong, thank you for your kindness. The paper just mentioned using IGF-1 to induce differentiation, but not maintenance, that is why I asked how to maintain the differentiated sy5y. Anyway, thanks. 

Here are some exemples for PC12 but I recommend you order the primers for at least 3 of them and check their expression under your conditions.

In order to achieve a good transfection cells need some space from each other (from 40 to 80% confluency). If you have too few cells it will cause your culture to grow poorly without cell-to-cell contact. If you have too many cells it results in contact inhibition, making cells resistant to uptake of foreign DNA. Actively dividing cells take up introduced DNA better than quiescent cells.

I would think that slew rate places a "limit" on the slope of the change that the output is expected to produce. So it does not depend on the input amplitude. If a step is applied to a first order network, the output changes and this change is limited in time by the amplifier characteristics. If the amplifier has a 10khz bandwidth, the output should change to 63% of the input step in one time constant, i.e is 15.9uS. If the slew rate permits this, then such an amplitude is allowable. if the slew rate is say .5V/uS, and we have an output change of 10V, then it will require 6.3*/.5= 12.6uS to reach 6.3V. One can even give a step of 15.9/1.26=12.62V and expect that slew rate does not place a limitation for the change. However if slew rate is .05V/uS, then even a step of more than 1.262V will get limited in the change by slew rate. A step of 1V is ok and a step of 2V is not. One has to consider the slew rate as the "LIMIT" on the change expected. A sinusoid has a max rate of change at 0 and will rise to 1.57 times the peak in quarter period. A 10khz sine, 10v peak will require a slope of at least 15.7V/25uS. or .628V/uS. So with a limited slew rate of .5V/uS, the sinusoid will tend to become triangular. If the amplitude is reduced to 7.96V peak, the slew rate will not "LIMIT" the rate of change of output.

A New in Vitro Model for the Study of Pancreatic A and B Cells

Pipeleers et al. Endocrinology 1985

This is an old method but our lab still uses it today to purify beta cells with a good yield and viability