Cell Differentiation - Science topic

Hi everone,

I need to use human M-CSF(100ng/ml) to induce mononuclear cells differentiation into macrophage.

But the issue is,should be use human serum or fetal bovine serum.

Thanks for your answer!

The goal is to check iNeural crest cell differentiation.

Hi,

I recently began working with THP-1 cells from ATCC and have followed the manufacturer's protocol, incorporating 2-mercaptoethanol and 10% heat-inactivated FBS. I am using RPMI from Thermo (not the ATCC version, Catalog number A1049101).

After three days of thawing the cells, their morphology appeared consistent with the ATCC guidelines (Fig 1). However, I noticed a change in phenotype, with the cells starting to adhere to the 75-flask.

I split the cells in suspension to a 6 well plate to allow a faster growing (also added a new media to the first flask to follow up the behavior). The cells in the flask have begun to elongate and firmly adhere to the bottom (Fig 2), while those in the 6-well plate are also attaching but remain in suspension, but showing a slow growth (Fig 3).

There is any suggestions for achieving a more optimal THP-1 expansion culture that I can subsequently differentiate to M0 using PMA? Thank you!

Hi,

I'm trying to differentiate iPSCs to Immune cells and looking for plates with low-attachment. I came across this reagent "Anti-Adherence Rinsing Solution" by stemcell technologies which is a surfactant solution for pre-treating cultureware to reduce surface tension and prevent cell adhesion.

But, my question is: can I use this solution on any TC treated plates or should it be only from aggrewell brand? Has anyone used it before?

https://www.stemcell.com/products/aggrewell-rinsing-solution.html Catalog #07010.

Thanks.

Vertica

The rate of glucose consumption by the neocortex is reduced by over 80% during anesthesia (Sibson et al. 1998), which disables the synapses (Richards 2002) that are supported by glial tissue (Engl and Attwell 2015). It is the synapses that provide the brain with its computational power (Hebb 1949). Disconnected (pig) neurons on life support (e.g., Sestan 2018)[1] have no ability to transfer information, and some might argue that such cells have been reduced to having a computational power below that of a single-cell organism, the amoeba (Saigusa et al. 2008), since they have been taken out of their ‘social’ environment for the expected programming between individual neural members. The organizational life of a multicellular organism is no trivial matter, requiring that each cell be subjected to some biological constraints (Albert et al. 2002) in exchange for the energy efficiency obtained per cell, which scales as the 3/4th power of an organism’s body mass (DeLong et al. 2010; Kleiber 1947; Wells 2007). This process has been shaped by 500 million years of evolution. We are a long way away from merely dumping a bunch of disconnected neurons into a dish that self-organize into a superorganism that supports consciousness, a well-studied topic by entomologist E.O. Wilson (Wilson 2012). Indeed, this calls for taking evolution seriously so that one day we might be able to really engineer a superorganism, which is not trivial (also see ‘converting rodents into humans’[2]).

Footnotes:

[1] The Yale researcher, Prof. Nenad Sestan, has managed to keep pig brains that were detached from the body and on life support (i.e. a warm blood supply mediated by pumps) alive for up to 36 hours (Regalado 2018). This result created quite a sensation at the National Institutes of Health with some even suggesting that this could yield the possibility of studying consciousness and the brain in the absence of the body. A notable observation was that the EEG activity of the pig brains was flat. What is clear from this is that a body is necessary to give the brain life through feedback (Tehovnik and Chen 2015). The challenge now is to determine how much of the body (or its prosthetic equivalent) is sufficient to provide function to a brain. A similar misthinking, as that which motivated Sestan (2018), has occurred by investigators who have hooked up two brains via wires to create the illusion that significant information can be transmitted between them (cf. Pais-Vieira et al. 2013 & Tehovnik and Teixeira e Silva 2014).

[2] Converting rodents into humans: Brain tissues from humans, called organoids, have been implanted into the brains of mice raising the possibility of having human brain cells incorporated into the rodent biostructure (Mansour et al. 2018). Some have speculated that this could endow rodents with an enhanced cognitive ability if the number of human cells were numerous enough (Begley 2017). Note that there are some 71 million neurons in a mouse brain, so this would require a significant addition of organized tissue. A fear persists amongst bioethicists that such implants might give rodents some degree of humanness: i.e., augmented consciousness. But injections of neural tissue into a foreign body are riddled with incompatibilities such as problems with blood supply, immunity, and functional connectivity.

The bird brain, unlike the human brain, regularly injects itself with new neurons via neurogenesis, and therefore it might provide clues about the challenges of adding new neurons into another’s nervous system (Barnea and Pravosudov 2011). Cell proliferation, cell migration via glia, and cell replacement are some of the steps that make up neurogenesis. Riding the brain of old cells is also part of the process (as anyone who has ever received chemotherapy understands). To add, this process is tightly regulated. For example, in the canary, neural augmentation occurs in the vocal control nuclei during periods of song and mating (Goldman and Nottebohm 1983). The point behind emphasizing this detail is to show that for the new neurons to contribute, one might need to reprogram the existing tissues—neurons, glia, epithelia—so that the new neurons are accepted and utilized effectively. At this point, injections of human neurons into a rodent brain may be more prone to producing cognitive deficits than cognitive enhancements.

Hi everyone,

I've been differentiating cardiomyocytes from iPSCs using stem cell technologies' ventricular cardiomyocyte differentiation kit. However, I've recently encountered a significant reduction in differentiation efficiency. I initiate differentiation 2-3 days after seeding, once the cells reach 80-90% confluency. I always use fresh media and supplements. I also tried differentiating cardiomyocytes from two different cell lines, but the outcome was similar. I noticed an increase in the non-CM population, cells appear degenerated around D6-D8 of differentiation and the cells don't start beating as expected. Can anyone give me advice on the subject? Any suggestions you can share would be greatly appreciated.

Thank you

Hi all,

Currently, I am trying to implement the Bibel et al. 2007 differentiation protocol from E14TG2a feeder-independent murine embryonic stem cells and facing some issues, primarily concerning impurities/neuronal density within the cultures.

Q1 - what are those cells with rigid cobblestone-like morphology alongside neurons?

Q2 - is it okay that my glutamatergic neurons cluster like that?

On day 8, I dissociate embryoid bodies into single cells and seed NPCs at 1.9x10^5 cells/cm2 on PLO/laminin-coated plates in N2 medium (DMEM/F12, 2 mM L-glutamine, 0.5x PenStrep, 1x N2 supplement and 50 μg/mL BSA), change medium after 2 hours and 1 day. Then on day10, I switch to B27 medium (DMEM/F12, 2 mM L-glutamine, 0.5x PenStrep, 1x B27 supplement).

I am working of LUHMES cells lines. I differentiate them for live cell calcium imaging. On the day of imaging, I stained the with fluo4 in differentiating medium (pH=7.4) and incubate the cells at 37C.

I am acquiring images at 20x, time interval of 1s and sometimes 2s, exposure time (100-150ms) for 30 minutes.

After 5 minutes of acquisition, I stimulate them with 2uM of ATP.

When I analyse the images, I dont observe spikes, in the cells and all the cells behave differently. In most of the cells I dont observe oscillations but increasing concentration of calcium. I also try to do imaging in saline buffer and dpbs but apparently cells did not like it and were stressed even before starting imaging.

Hi,

I am working with the U937 cell line, which I wish to use for macrophage survival assays. For my protocol I dilute my cells to 0.5x106 cells per ml (or well) and add PMA at a concentration of 100ng/ml for 24 hours. The cells are then washed with PBS and then serum-free media (RPMI with penicillin/streptomycin) is added for 48 hours before bacteria is added at a MOI of 5:1 (bacteria: differentiated U937s) and I carry out the assay procedure.

However, I'm finding that my differentiated U937s are not adhering to the bottom of the wells and do not have characteristic differentiated-macropage-like cells after the PMA and 48 hours in serum-free medium. They are easily washed off with PBS.

I am not sure why this is happening. Another colleague has had no problem with the U937s.

I am wondering if the U937s are too high a passage number (p38)?

Or if I should try carrying the PMA concentration or some other aspect of my protocol? Any advice is appreciated!

Hello all

I'm working with SHSY5Y cells to differentiate them to neuronal cells. I'm following the protocol with using RA, BDNF and cAMP for cell differentiation. I tried it different times but it was unsuccessful. Can anyone one help me to do this successfully?

Thank you

I have THP-1 cells differentiated using 20ng/ml PMA and incubated in 12-well plates at 2x10^5. I would like to lyse the cells and extract LL-37 or hCAP18 from my cells to use for an human LL-37 ELISA kit.

I have zero experience with protien extraction so I'm on the deep end with this one. Reviewing literature and consulting with people that have worked on protien extraction has led me in several directions for possible approaches.

I have gotten recommendations to use RIPA lysis buffer, UREA lysis buffer or just cooled PBS for the extraction. I've been recommended to use repeated freeze-thawing or a cell distruptor.

Do I need to use SDS page(gel) to confirm the presence of the protien in solution?

Easy to say I could really use some guidance from anyone that has experience with this particular extraction and ELISA procedure.

Much appreciated.

I followed an immunofluorescence staining protocol for my cells differentiated into neurons at D14. Using 4% PFA (Alfa Aesar product) for fixation in PBS, I observed that 2-3 out of 8 wells were empty or neurons were drift towards one side of the well after D1. Upon completing the protocol and examining under a fluorescence microscope, I noticed 7 out of 8 wells were empty, with one exhibiting perfect fixation and staining. What might be causing this issue?

I recently thawed a vial of THP-1 cells . The revival was low but I could see good number of cells growing. But two days later the cell population increased but a lot of cells settled down and now they are differentiated even without a stimulant. I don't see any turbidity in the media. No contamination. Could it be low media volume? Also I left the flask horizontal and not vertical.

I have been growing SH-SY5Y cell line in DMEM/F12 media supplemented with 15% heat-inactivated FBS. For differentiation, I plated 10000 cells per well in 96 well plates and changed media every day, which contained 3% FBS with 10 micromoles RA, and also I have tried with 10% FBS and 10 micromoles RA.

In 3% FBS, I noticed cell death within two days of differentiation, and in 10% FBS after five days the well was almost filled with cells. So what should be the ideal FBS concentration and seeding density for differentiation?

I have attached image of the SH-SY5Y cell line acquired after 5 days of differentiation with 10% FBS and 10 micromoles RA most of the cells look like epithelial

I have been having issues with my THP-1 cells for most of this year - when I try to differentiate them with PMA into macrophage-like cells about 1/3 of the cells do not adhere properly (either floating or loosely attached) and most of these seem to die. I have tried to wash these cells off and use the ones that are adhered down but during experiments these are not happy and will round up and de-attach.

I have tried everything I can think of - gotten new PMA, tried different PMA concentrations, all new media and FBS, tried different cell densities, different culture plates etc. but nothing seems to make a difference. The cells are also negative for mycoplasma. I have experienced this issue with different batches of THP-1s, including a recently ordered batch from ATCC, which suggests it could be the way I am culturing the cells but I have not changed anything from the way I normally culture them - and they have been fine for 2 years prior to this issue.

I culture the cells in RPMI + L-glut + 10% FBS. I typically use 50 ng/ml PMA for 3 days and then an overnight rest before experiments (but I see this issue within 24hrs of PMA addition). My monocyte cultures do not exceed 1x10^6 cells/ml prior to seeding.

Has anyone come across this issue before?

Thanks in advance for any help with this.

Hello everyone,

We needed cardiac cells for a project and obtained H9C2 rat cardiovascular myoblast cells from another laboratory. The cells are at passage 15 and unfortunately have differentiated into cardiomyotubes before. Can this differentiation be simply reversed? Is there anything we can do?

Thank you very much.

My research project aims to investigate the effects of bilirubin on the maturation and function of fibroblasts, I was going to differentiate mesenchymal stem cells into fibroblasts to then investigate. However, I have now found current studies to describe the two as indistinguishable, this is including their morphology, gene expression patterns, surface markers, proliferation, differentiation, and immunomodulatory capacities. Any advice is much appreciated.

I am trying to differentiate H9c2 cells into cardiomyocytes using 10 nM all-trans-retinoic acid. The cell growth seemed to be decreased after treatment with 10 nM RA. I plated H9c2 cells on T75 (35K/ml) and treated with 10 nM RA containing 1% FBS.

I have two questions.

Q1. Does the cell grow after differentiation or do I have to differentiate everytime to run the experiment?

Q2. Can I switch back to 10% FBS after the cell differentiate or do I need to treat 10 nM RA daily?

I am passaging my hiPSCs with EDTA and E8 medium. However, I have noticed the presence of spontaneously differentiated cells in the final passage.

What other techniques are available to improve the purity of the final passage, and is there any literature on this?

Hello All,

I'm working on SHSY5Y cells differentiation. My problems are:

1-After adding the final differentiation media to the cell cultures, they started to detach from the plates, and became floating day by day. However, I've coated the plates previously by Poly-D-Lysin.

2- After adding the final differentiation media, cells look clumped.

Can someone suggest me differentiation protocol or any suggestion to solve my problems.

Thanks in advance.

For analysis of C2C12 myoblast cell differentiation in Cell Profiler Software.To assess Myosin Heavy Chain expression in differentiated C2C12 cells.

I have cultured N2A cells and I'm trying to induce them to differentiate into neurons using retinoic acid. Could anyone recommend any protocols for inducing N2A cells differentiation into neurons using retinoic acid, with specific quantities of time, concentration and number of cells?

hi everyone, I am working on differentiation of fibroblasts to iPSCs. I am planning to. use matrigel coated plates, but am a little lost on what concentration I should use. I am using Corning matrigel (REF 356234). The batch I have has a protein concentration of 8.9 mg/mL. the data sheet just says to not dilute to less than 3 mg/mL. in your experience what isna good concentration to use for this application?

Hello,

I am looking for a protocol to differentiate dendritic cells from monocytes.

I would like to isolate monocytes from PBMCs using the EasySep™ Human Monocyte Isolation Kit (StemCell).

I found information that DC can be obtained by culturing monocytes with IL-4 and GM-CSF for several days. Various cytokine concentrations and culture times are reported in the publications. Has anyone differentiated DCs this way and has a good protocol?

Or are you using the ImmunoCult™ (StemCell) dendritic cell culture kit?

We are analysing the expression of Afp (endoderm marker), Sma (mesoderm) and Tuj1 (ectoderm) by mESCs that have been cultured in spontaneous differentiation conditions for 10 days with immunofluorescence staining, and we are detecting tha signal for more than one marker on several cells at the same time, i.e. we observe cells that are positive for Afp and for Tuj1. Is it possible that a single cell expresses more than one of these markers at once?

I would like to perform real time study on the effect on various differentiation conditions on the collagens production in MSCs during differentiation into chondrocytes. I would like to visualize it via fluorescence microscopy (with the specific fluorescence microscope placed in the incubator). Is it possible to stain collagens in living cells (without fixation and permeabilization)?

Many thanks in advance.

I differentiate N2a cells with 20 uM retinoic acid 1% serum in a 24 well plate. During the differentiation process that lasts 4 days, I change the medium every two days, but on the 4th day the cells start to die. How can i make cells live for a long time (10 days)?

Hello everyone.

I am going to analyze differentiated cardiomyocytes (CM) via confocal microscopy. However, I struggle to attach CM properly to the glass slide/plate. Although I coated the glass slides with LM-E8, the CM could not attach firmly and started to detach after adding 4% PFA to cell staining.

Does any appropriate coating for CM culture over the glass?

I appreciate any recommendations and comments.

Best,

Fatemeh.

Hello everyone,

I am using retinoic acid to differentiate SH-SY5Y cells into neuronal cells to generate a Parkinson's disease model. I will perform MTT assay before using a neurotoxin of interest to generate a PD model again for another set of experiments but I am not sure if I have to differentiate the cells again because I have seen that the same cell line is used undifferentiated as well, for example in Alzheimer's disease research.

Is it a "must" for in vitro PD model?

Thank you

I would like to generate some iPSC cell lines from T1D donors and matched controls. I am interested in re-programming kits that work well with fibroblasts and are non-integrative, and preferably non-viral. The downstream beta cell differentiation is complicated and difficult enough that I don't want to also have to heavily troubleshoot the iPSC generation. So, the kits, though expensive, are very attractive to me. Are there any kits that are better than others for fibroblasts? Any kits you've used that you suggest avoiding for one reason or another? TIA

Dear all,

Do you have the experience in MSC chondrogenic differentiation and immunostaining at the specified time points?

I used to work with adherent cells only so I feel a little uncertain. As I understand from the literature, differentiated MSCs form aggregates and start to float in medium. Is that true? If so, how does one fix, stain, and then visualize the aggregates? Can I do it without the access to the mictrotome and paraffin embedding etc.? As far as I understand, the aggregates might have even 400 um in diameter so quite large objects.

Thank you.

Hello! I currently have differentiated iPSCs I wish to clean up. I use mTESR with matrigel coating.

ReLeSR selectively dissociates healthy stem cells, however even on quick release (1 minute), all the cells immediately dissociate, and I am unable to isolate healthy stem cells.

I think that it might be due to me passaging very regularly (once every three days), and the cells have been adapted and now dissociate easily.

I would also like to attribute that to the reason why my iPSCs are differentiated. I supplement them with ROCK inhibitor (5uM) every time I passage. Given that they are passaged once every three days, that means they are in ROCK inhibitor once every two days... could this be the reason my iPSCs are so differentiated, as I did not give them sufficient time to recover from ROCK inhibitor?

However, the reason why I passage so regularly is because I know need to passage once I see differentiated cells... Do you suggest I completely omit ROCK inhibitor during passaging, and would it help the maintenance of my iPSCs? For individuals with experience using ROCK inhibitor during passaging, do you feel that frequent passaging causes spontaneous differentiation of iPSCs even when you remove ROCK inhibitor the next day (before 24 hours)?

I would really appreciate any advice that would help me out of this predicament, thank you so much!

Hi folks,

I extracted BAL fluid from mice to check total and differential leukocyte counts, as well as inflammatory cytokines and chemokines, and now I'm wondering how long I can keep my samples at -80 degrees to check the same. Suggest some practical experiences or methods for a better result.

I am interested in studying targeted cancer immunotherapy, particularly, I want to prevent the tumourigenesis of the cells. I would like to know if there is any genes or promoters that activate differently than normal somatic cells.

I recognise the TERT gene that will activate once the cells are highly proliferative, but it also expresses in the early stage of cell differentiation process as well. So, I need to find others instead.

I think my cells have differentiated but I'm not sure.

How can I be sure of the identity of my cells?

My project depends on the accuracy of this cell line.

Hello,

I am not sure if my ipsc cells' morphology look normal. Can anybody tell me?

Thank you!

Franklin

I want to know after generating Knockout (KO) cell line of Human embryonic stem cell, how we can differentiate cell based on cell quality. Any specific protocol available which leads the specific results?

Dear Colleagues

I am going to do cell differentiation of pre-osteoblast to mature osteoblast by giving induction from my materials.

Is that possible to grow the pre-osteoblasts cell line (MC3T3-E1 Subclone 4 CRL-2593) in the DMEM medium (product code LM 001-05). We have this medium in our lab. However, I saw that a lot of papers used a variety of MEM alpha (recommended by ATCC is MEM alpha without ascorbic acid for base medium, product code A1049001).

If I should use MEM alpha, could you kindly recommend what kind of MEM alpha should I use, please? Do I need to use MEM alpha included ascorbic acid or not? In our lab we have L-ascorbic acid separately (product code 255564).

Thank you and best regards.

Hi everyone,

We are currently doing C2C12 cell differentiation and unfortunately, even after 4 days in the differentiation medium, the cells elongated but we do not see cell fusion at all. We stained cells with MyHC and all the cells are MyHC positive and single nucleated.

We used just a simple protocol for differentiation with DMEM, 2% heat-inactivated horse serum, 1% PenStrep, and the medium is refreshed every other day. The C2C12 was from the other lab and we don't know which passage they were in.

Could anyone suggest the possibilities that can happen to affect C2C12 cell fusion to form myotubes?

My SHSY-5Y cell line morphology is changing on it's own and getting a morphology that resembles differentiated cell line. Could anyone provide me an insight of what is happening.

I am also attaching a picture of the cell line here with.

By a 'higher differentiation capacity' I mean that it would be easier to differentiate them– especially without adding a pool of chemical factors.

I think cell lines might be tricky as they are somehow modified but stem cells (MSC, iPSC) might be more potent as they are there to differentiate.

Any insight is welcomed!

Hello,

I am trying to differentiate HEK293 cells to induce ciliogenesis. I have seen in papers that this is possible by serum starvation but I obtain a mix population of differentiated cells and many dividing cells.

Thank you very much in advance!

Dear all,

I am differentiating BM cells into BMDM using L929 conditioned media. Briefly, at day 0, I isolate BM cells from mice and seed it to 100pi petri dish using complete with 30% LCM (BMDM media). At day 1, i collect non-adherent cells and seed it (1x10^6 per dish) to 100pi culture dish using BMDM media. And i add 10ml of fresh BMDM media at day 4 and change half of this media to fresh BMDM media at day 7.

In this process, my cells differentiate too much and reach 100% confluency at day 5~6. Non-differentiated cells floats or attached lightly above the monolayer of differentiated cells. I wash them with 1X PBS before detachment of BMDM to eliminate them, but is it okay to just run the experiments? I'm afraid that i may have done something wrong during culture.

Also, morphology of differentiated cells are different with other reference images. Mine is more attached to the dish than other images, and have morphologies close to round shape with several short branches like LPS-stimulated cells. It also has something looks like vacuoles. I attached images which is similar with my cells.

I want some clues for my questions.

Thank you for reading.

I am characterizing differentiated cells derived from a specific hPSc line. The idea is to compare the differentiated cells in "Semi-matured" state to a later "matured" state. I would like to know, whether I should use the gating applied on "Semi-matured" cells to "matured" cells as well, or should I use different different gating for these two populations?

We are doing studies on B cell differentiation and would like to expand human B cells in vitro.

Thank you

I differentiated MC3T3-E1 cells to osteoblasts by using differentiation kit. I put some vials of differentiated cells in nitrogen tank. Recently, I used 2 vials of same passage number and I saw these little things only in one of my flask, and the other flask was fine.

I put cells from both flasks on my biomaterials and did XTT, and there were no significant difference between data!

Does anyone see anything similar to these in their culture medium?

We are trying to differentiate SH-SY5Y human neuroblastoma cells to individual dopaminergic nerve cells by using retinoic acid. The protocol takes 6 days to complete but our cells form clusters during day 4-5 and since the cells at the top can't use enough medium they start to die.

What could be done to make those cells grow as individuals and see the connections between the neighboring cells?

Hi,

I applied on PC12 cells 100 ng/ml NGF and incubated for 72 hours to differentiation. After 72 h, approximately most cells differentiate and then I applied an anticancer drug but all of the cells including the control group died. The medium I used while applying the drug is the same as the medium I used for differentiation. I couldn't find my mistake. I'm waiting your idea or advice.

I'm having an issue with differentiaon media (DM) of MC3T3-E1 Subclone 4 cells I am following the ATCC recommendations for media and supplements (alpha-MEM + 10% FBS + 1X anti/anti (Pen/strep/amphotericinB)) and culture conditions for revival.

I use using DM composition- 10mM BGP; 100ug/ml L-ascorbic acid; 10uM Dexamethasone, but there is debris formation and cells are not looking healthy

Any suggestions as to what might be the problem or recommendations for the same?

Below I have attached the pic of cells after DM 4days

I have been having difficulty differentiating C2C12 cells into myotubes. I use DMEM media with high glucose + glutamine + sodium pyruvate supplemented with 2% horse serum and 1% penicillin/streptomycin. However, I have noticed most published work on C2C12 cell line use DMEM high glucose + glutamine, no sodium pyruvate. Could the presence of sodium pyruvate in my media be affecting my differentiation?

Is sodium pyruvate not suitable for C2C12 cell lines?

I'm working with H9C2 cells for the first time and I've been following protocols I could find on papers and research gate. For the sub-culture, I seed 10k cells/cm2 and split the cells every 2-3 days as needed. I've then continued with a differentiation protocol in which I added 1 uM or 10 nM RA in dark daily for 5-7 days, and measured cardiomyogenic markers such as Actc1, Myl2, and Tnnt2. However, I saw no difference in gene expression (qPCR) in RA-treated cells compared to DMSO-treated although there were morphological differences. For the differentiation experiment, I seeded 30k cells/cm2 on a 12-well plate.

Is there anything I'm doing not right? For example too high seeding density? Can anyone experienced with handling H9C2 cells please help?

Hi everyone,

I just differentiated my neuroblastoma SK-N-SH cells with 10µM Retinoic Acid in 6 days and I got the pictures of non-treated and treated cells below. Do you (especially neuron experts) see any differences in the morphology of the cells?

Thank you so much!

HI everyone! I am going to start culturing RPTEC/hTERT renal proximal epitelial cells and I have several questions about it. First of all I want to know when the cells are proliferating and when differentiating. I have cultured immortalized podocytes previously and these cells proliferate at 33ºC and differentiate at 37ºC, so i have clearly differentiated the two populations. But in the case of RPTEC/htert cells I dont know how to know it, because I think they dont have the SV40 T antigen to thermoswitch them. So, are cells proliferating and differentaiting joint? How to know which are mature cells? And if I want to treat them with something, I will treat proliferating and differentiating cells?

Another question is the appropiate culture medium for these cells. I have read that the supplied medium from evercyte or ATCC in some cases didn't work very well, and as well the mixture of DMEM/HAMs F12 . So the best option is to buy DMEM (low glucose with glutamine?) and F12 separately, plus glutamax, EGF, ITS, Penicillim/Streptomycin and hydrocortisone? and FBS is necessary or not?? Because I have read the two options

I would appreciate any answer from peolple working with this cell line.

Thank you in advance

Ana

Dear everyone, 

I am currently trying to differentiate MGE-01 cells into platelets. Since I never worked with MEG-01 cells before, i run a pilot using these two paper as reference:

·         24h – pseudopodia (Yang et al., 2016) with 80 ng TPO

·         48h – platelet-like particles (Risitano et al., 2012)  with 100ng TPO

I seeded the cells in a 6 well plate with a cell density of 0.3 x 106 and added 100ng of TPO. I checked the cells every 6 or 12 hours and my results are: 

1.No pseudopodia appeared at 24h 

2.No proto-platelets appear at 48h

3. "Pseudopodia" appeared at 60h in my sample.

My questions are:

1- How long should it take to differentiate platelets adding 100 ng of TPO to the medium? Is there anybody using a higher concentration?

2- During the days of incubation should I add TPO everyday or just when I change the medium?

3- I attached two pictures at 60hours from when I added 100ng of TPO. I would like to you if the "spider like cells" that i see are megakaryocites with pseudopodia or something else. 

Every kind of help would be much appreciated.

I have read so many papers on vitD3 acting as differentiation agent. I tried this and found that I got differentiation only once when I freshly dissolved calcitriol in ethanol and thereafter I never observed even after increasing concentration. It's seems to be highly air sensitive. Do I need to make aliquots in anaerobic glove box? Or could you suggest me the right way to use it?

I am wondering if anyone has had success with in vitro polarization (via cytokines) of Jurkat cells. My goal is to understand the affect of a drug on Treg induction and differentiation. I understand that this would be reasonable using human/mouse primary T cells, however this may not be practical and or feasible in my case, at least for now. I would like to first explore the effect of this drug on T cell activation/differentiation using Jurkat cells. I am having trouble finding literature on this topic. Thanks in advance.

Our group is starting to work with BAL samples from mice. One of our aims is to analyze BAL cells by differential cell count. Many authors report doing a cytocentrifugation, but we don't have this equipment. Is there an alternative to cytocentrifugation? Does anyone have a protocol that can help us?

Hi,

What is the common B cell differentiation stage used for hybridoma technology? I understand that these are antibody producing B cells upon the injection of antigen, but do people use mature B cells or fully differentiated plasma cells to fuse with myeloma cells?

Or is the specific cell differentiation stage less of a concern?

Best,

Cece

Lately, I've been having a lot of difficulty differentiating L6 myoblasts and I'm unsure what I'm doing wrong.

After adding the cells to 6 well plates (200,000 cells per well) and allowing them to grow for 48 hours in growth medium (GM: AMEM supplemented with FBS and antibiotic-antimycotic agents to final concentrations of 10 and 1%, respectively), I wash with PBS and shift the cells to differentiation medium (DM: AMEM as above except that 2% HS replaced 10% FBS). Within 48 hours of differentiation, the majority of the cells die and the remaining cells differentiate very poorly such that no myotubes form by day 5 of differentiation.

The only thing that I can think of is that the wells are too confluent by the time that I transfer the cells to DM. Attached I've added a picture of a well just prior to transferring it to DM - you can see that it is nearly 100% confluent.

Any help would be greatly appreciated!

Does anyone know of studies investigating the optimal well sizes for growth/hypertrophy of C2C12 myoblast-derived myotubes following differentiation using standard serum starvation? I find that many papers don't report well-sizes for their experiments, however I tend to think that myotube differentiation and growth might be affected by well area, regardless of similar confluence level across different well sizes at the onset of differentiation. Any thoughts/experiences?

It's time to passage my iPS cells, so I add 1 mL Accutase per well for 5 min at room temperature (edge has folded back). But after aspirating Accutase and rinsing with fresh mTeSR1, I observed that undifferentiated cell colonies are still attaching.

In the below link, it mentioned that "differentiated cells are more strongly attached than undifferentiated cells", although I didn't find its reference source.

https://cellculturedish.com/release-your-cells-a-simple-new-tool-that-eliminates-the-need-for-manual-selection-and-scraping-in-hpsc-passaging/

My question are: (1) why differentiated cells release first after treated with Accutase? (2) whether I distinguish the cell differentiation correctly? Here, I regard fig 1 as differentiated cells, and fig 2 as undifferentiated cells.

Thanks for any answers or suggestions on this issue.

I am totally new to feeder-dependent iPSC culturing. Previously I was doing on feeder-free iPSC culturing. Hence I encountered that the iPSCs which grow on the feeder are sparse and not growing as typical colonies which are compacted. From the attached photos, under 4x magnification some cells are individually apart from each other while some are in close contact with each other. Under 10x magnification, those "individual" cells look big without a defined shape, I am not too sure are they cells differentiated or undergoing apoptosis. Before changing medium iPSCs look a bit spiky in shape, after changing, they are more round in shape and starting to form a slightly more round colonies. Can anyone with experiences provide me some advice on this. Thank you.

Hello everyone, I would like to start single-cell sequencing on differentiated cells from human iPSCs. I want to discuss here the more cost-effective way to obtain publication-quality data from the analysis.

If you have already experienced a certain device or method, please share the annual costs including the system itself, reagents, installation fee, training fee, software, and maintenance fee, etc.

Or should I simply send my samples to the sc-Seq service?

Thank you very much for your time and info in advance.

I did a bone marrow dendritic cell differentiation (7days) using Gm-csf at both day 0 and day 4,

the question is, Which CD is more specific marker to check ex-vivo Dendritic cell differentiation using flowcytometry?

i am wondering whether CD80, 86, 40, MHCII and CCR7 will be the best option?

or CD11c, CD123?

or there is a more specific marker to validate the differentiation?

I differentiated human neural progenitor cells into neurons. These cells were relatively fragile and were difficult to attach on the cell surface. I have to immunostain these cells, thus I was wondering if the temperature of PFA may affect the immunostaining results obtained from the cells.

I am doing a primary culture, and I have some problems while culturing the cells.

When I culture the cells from primary to passage 1 and 2...

lots of cells differentiate and stop dividing before I get a sufficient amount of cells.

How can I prevent primary cells from differentiating and divide well?

I am looking to know other people's experience with seeding C2C12 cells on 96 well plates. Preferably, I want to minimise the proliferation time but optimise the conditions for differentiation to occur, likely between 70-80% confluency. Thank you in advance for your assistance.

I am struggling to find information about this- I'm not sure if it is something so obvious that it is assumed rather than stated in reseach papers (!) or if it is just not the case, or maybe I am just looking in the wrong places!

I am trying out some differentiation protocols, and wondering if I can use a comparison of proliferation rates (for example with BrdU assay) between the 'control' stem cells (human foetal neural stem cells) and experimental cells to show they have differentiated

Any ideas gratefully received-

Thanks

In the literature I found this: "after 3 day post-confluency, cells are differentiated." But how? With or without FBS? If with, than 1, 5, 10% or changing? How to prevent outgrowing? Thanks

Dear,

I haved try to test the effect of some small molecules differentiate the aml cell line, including HL60 and PL21 cells, by using the PMA as positive control. CD14 and CD11b were measured for charachterization of differentiated cell (monocyte/macrophage).

My results revealed that the PMA-induced group express both of CD14 and CD11b. However, the small molecules-treated group expressed only CD14 (higher than PMA group), but not CD11b.

I wonder to explain the results but not sure what is cell type that express CD14 but not CD11b.

Thank you

Best,

Sarit

I have been trying to generate RPE from iPSCs. Though I'm able to get the cobblestone morphology in the differentiated cells, they are not getting well pigmented. Please share your insights on the same.

Thank you.