Questions related to Stem Cell Culture
In my upcoming project, I will use poly-L-lysine coated culture flasks due to the culture conditions of neural stem cells.
One thing that comes to my mind is if I need to perform a viability test such as MTT or SRB on these cells, do the 96-well plates have to be poly-L-lysine coated as well? Buying manufactured coated plates increases the cost a lot. I am guessing my deviations may increase if I coat them myself. Also, does poly-L-lysine interfere with these kinds of tests? I do not have much experience with this subject. I would be very happy if you share your information not only for the viability tests but also for any kind of plate-based tests for the neural stem cells.
Thank you very much.
Hallo! Does someone have experience culturing cells derived from infectious samples?
I would like to isolate the bone marrow mesenchymal stem cells from patients diagnosed with bone infection and culture them. The only problem is that the tissue would be infectious with bacteria inside. Some of the bacteria might be also multiresistant. If I culture them in the incubator, there might generate bacteria membrane, and even contaminate other cell wells. Is there any good idea how I can eliminate these bacteria and culture these stem cells?
We are experiencing a significant amount of cell death upon differentiation of h9 hESCs to definitive endoderm cells. We grow h9 cells in feeder free conditions and induce differentiation to endoderm using RPMI 1640 supplemented with 2mM glutamine, 1X NEAA, 100ng/ml Activin A, and .1% B27. After 1 day of induction we add .1X ITS, after 2 days induction we add 1x ITS. We are trying to perform an initial RT-qPCR batch assay on day 3 cells, however by that time we have only a few cells left with endodermal morphology - most of our initial stem cell culture has lifted off the plate and died. Does anyone have any successful strategies for increasing cell count at day 3, while maintaining a relatively high degree of differentiation? Would greatly appreciate any insight - Thanks!
So, I will be culturing H1 over geltrex, which will be subjected to electroporation, and subsequently FACS-single cell sorting.
I am currently deciding which of these two medium shall I work with..., any suggestions will be very much appreciated! Thanks!!!!!!
I am doing fibroblast, stem cell culture for sequencing. (From primary culture to the end)
Since FBS is expensive, there are some protocols that mix FBS and horse serum (1:1)
I have done mixing them, and culture results were not that different. They grew well like in normal FBS containing medium.
But I have to do WGS with the cells I grew, so I am suspicious about the result.
Even if the growth rate and cell morphologies are the same, do you think the DNA sequencing results are different when the FBS is mixed with other serum?
I am growing organoids in monolayer but honestly couldn't manage to make it confluent. the protocol i followed made use of collagen. they first look "like confluent" with about 70-80% cell, but after washing or changing media, they just turn from 40 to 10% remaining cells.
i thought there could be a problem with collagen? what is the best collagen concentration and how much collagen should be used to coat per well? Thank you.
If we seed human pluripotent stem cells (hPSCs) in absolute Matrigel and allow them to form embryoid bodies over the next ~5 days then how much time it takes for them to spontaneously differentiate when we remove the stem cell culture media and maintain them in spontaneous differentiation media? Also, I'd like to know different media for spontaneous differentiation.
Please let me know if you have any information regarding this or if you've come across any articles or know anybody in this field who can help me.
Thanks in advance!
I've been working with some bone marrow derived MSCs I got from Texas A&M and I've wondered what other's experience has been with bone marrow derived MSCs (or MSCs from adipose tissue, hUCB etc. doesn't matter). In my experience they tend to be slow growers (plated at ~2000 cells/cm^2 it'll take ~10 days to reach 80% confluency) and senesce quickly (I get like 7-10 passages out of them). I change the media weekly and don't use a pH indicator. I've been using stemlife MSC medium for undifferentiated expansion. This media is interesting to me because I assume its probably pretty basic but it contains what they call "life-factors" which is proprietary of course.
Has anyone on here created their own media or found a particular method of culturing to be the most robust and productive when working with bone marrow derived MSC's?
March 18, 2020: COVID-19: Why can't we ramp up production of gamma globulin from recovered individuals, to treat those in imminent danger of Covid-19 morbidity and mortality? Same question for production from Stem Cell labs, using those from recovered patients, in the manner that we used to us, for example, ALF to treat infections in immune compromised patients.
Does anyone know that how many cells lines and primary culture can be grown in L-15 medium? I need the complete list of cell lines and tissue types.
Our CO2 incubator in cell culture facility is not working and we need to grow our cells in L-15 medium which does not require CO2 for buffering. Another medium is CO2 independent medium by Thermofisher but the information about this medium is quite scarce.
We are trying to run a blot for LC3 [MAP1LC3B], however unlike the two 14 & 16 KD band we are getting only one band (picture attached). This blot has 5 treatments: Lane1: Control, Lane2-5: Treatment in increasing order Cell used for this expt: HEK293 Gel % = 10% in 1mm spacer glass Development: Manually in Dark Room on X-ray film Primary Antibody dilution= 1: 1000 (as recommended), overnight treatment at 4C Secondary Antibody dilution= 1;10,000 (recommended= 1;10,000 to 20,000) 1 hr at RT Chemi-luminescent: ECL plus 1:1 in 1 ml H2O Please kindly assist why we are getting only one band in all the samples. Also why all the samples seems to be attached with each an other and why they appear zig-zag is structure rather than straight
I have tried to weigh out lipolyzed collagenase to be used for stem cell culture, but it seems to either fall off- or blow off the sides of the spatula inside the fume hood where we measure out our chemicals. Does anyone have any techniques to reduce losing so much of the samples? I am using a very small and thin spatula, so that I am able to reach down inside the container and place 10 mg of collagenase into a 15 mL tube.
I was freezing Wharton Jelly stem cell in Mr.Frosty and forgot them in -20 degree for 48hours (due to weekend). I transferred them to -80 degree in Monday morning. Does anyone has experienced this?
Hello, I have stem cells in culture in a plastic flask and I want to observe it in a Scanning Electron Microscope (SEM), i want to know if there is a special preparation to the culture whitout using glass slides or scaffolds?
We are doing fruit stem cell culture and need to keep some of our cultures going for up to 1 months. For one week, we have struggled with yeast contamination in our cultures. However, we wear gloves when handling cultures and media. Yet we continue yeast in our cultures. Is it possible to clear the yeast contamination?
I went through a basic protocol provided by Brewer et al., 1994
1. The media was stored according to instruction (-4 to -20 for B27, -20 for trypsin and 2-8C for neurobasal and 15C for Glutamax)
2. The hippocampus of mature rat (6W) was extracted under the sterile condition
3. The extracted hippocampus was stored in HANK and Pen/Step for 2 min
4. The hippocampus was homogenized for 15min using trypsin-EDTA 0.05 (using micropipette with blue and yellow tips). i had extrac care about buble formation
5. The trypsin was inactivated and centrifuged, the pellet was re-suspended in 500ul hank+pen
6. The cells was counted (2 * 10E6) and inoculated into the culture media (5ml neurobasal+ 2% b27+glutamax 1%) in a T25 flask (adherent + filter cap)
7. The cells were incubated at 37C and 5% Co2
* No antibiotic in culture medium / even addition of antibiotic dosen't make change. Furthermore, i didnt have contamination
* No serum in the medium
* No bFGF/ even addition of bFGF dosen't make change
Still i have some growth that i don't know what they are. I think they are not typical neural cells. In my though, the problem is that the cells are die within few days.
what went wrong?
I am using the TeSR-E8 medium for the maintenance and growth of iPSCs. I made the aliquotes of complete medium without adding Pen/Strp and Normocine, and stored at -20C. On need, I thaw one of them and store the remaining volume at 4C where it remains free of contamination for one week. But it gets contaminated after that even though I use and store the vial very carefully. Can I please get any suggestions regarding the use and store of stem cell medium? Is it ok for iPSCs to use medium containing Pen/Str and Normocine?
I wish to induce stress conditions to Adipose derived mesenchymal stem cells and for that i have these three available chemicals. Your expertise are needed.
Update: Cells are growing fine in our lab now!
I'm the only person in my lab starting on iPSc culture (we received a vial from another lab and all I have is some the medium they use for culture) and I'd like some tips and help. I have human iPS cells, and I culture in mTeSR1 on matrigel coated plates (so feeder free), replacing media daily. I try to remove what I think is differentiation by marking on the plate while looking through a microcscope. One cell line shows nice colonies like I saw on suppliers webpages (Like Stemcell or Invitrogen), the other I'm not so sure...
I've attached some pictures, can someone tell me if this is what it's supposed to look like? And can give someone tips on how to passage? I've passaged one with gently scraping, like i saw mentioned in literature and tried picking the colonies with a p1000 tip (but didn't know if I actually picked up a colony or not, so I tried scraping). Maybe i just need a bit more patience :)
While awesome to start things up, it's frustrating having no one to ask if things are supposed to be this way :)
Which concentration of Matrigel you use for iPSC culture? I was using 500 ul in 15ml and then, I have added 2 ml in 6 cm dish. However, my colonies did not attach.
Now, I am using 1.5ml of matrigel in 12 ml and so far, nothing.
I am adding (EGTA 2mM-10mM) to my mouse neural stem cell culture (media is made up of DMEM +10%FBS + 5% Horse Serum + 1% Sodium Pyruvate). I see the media changing color to orange, and the cells are rounding up and dying. However, when I PH the media has not changed in PH. What else could be causing this effect?
Currently I am working on mechanism of astrocyte development. For this I am making neural stem cell cultures (mouse).I am using CRISPR/Cas9 strategy to knock out genes. My plasmid is puromycin resistant. So I am planing to do Puromycin titration for my experiment. But I don't have a standard protocol for puromycin treatment. can anyone help me.
Real-Time is done and Mesenchymal stem cells had expression of cTNT, CTNN1 and other Cardiomyocyte cells markers
is it possible or cTNT is an specific Cardiomyocyte marker and something wrong is in this result ?
I am trying to transfect canine Ad-MSCs and NSCs with Bmi-1 plasmid by using lipofectamine 3000, but every time i do the transfected cells start dying after 48hrs, i am using following method for 6-well plate
· Per well
1. 125µL opti MEM + 5µL lipofectamine
2. 125µL opti MEM + 10µg P3000 + 5µg DNA
3. Mix both 1 & 2 and incubation for 20 min then put in cell culture drop by drop
4. after 6 hr serum & antibiotic free culture media is changed to full culture media
the first picture is of cell culture after 24-hrs and 2nd is after 48-hr
RWPE-1 and WPE-stem are two prostate cell lines which need K-SFM(Gibco 17005-042) as their culture medium,which is in the protocol provided by ATCC.Problem is that in China mainland,because the medium has BPE(bovine pituitary extract) as a component,it is prohibited to import from overseas.My colleagues told me KM(sciencell 2101) or defined K-SFM(Gibco 10744-019) could be used as equivalence.But I asked technical support of Gibco to assure it,they gave me a negative answer.Could anyone tell me is there substitute medium which also could be used to culture these two cells? Thanks!
Could you recommend is the best kit for isolation of CD34+ cells from bone marrow samples, using beads magnetic?
I'm an undergraduate researcher at UCLA and I was assigned to start a stem cell culture and differentiate those cells into cardiomyocytes. Currently using the GiWi method (CHIR and IWR1). I find that a large number of my xc-HUF-1 cells begin to die after Mesoderm induction by CHIR occurs after D0 (2 days after single cell seeding). In contrast, the H9 cells that I'm also differentiating seem to have minimal cell death after CHIR addition. I'm currently in the process of modifying the protocol using varying amounts of CHIR concentration and hopefully I'll be able to obtain beating cardiomyocytes. For the time being, I was wondering if any of you all have any advice / suggestions regarding my differentiation protocol. I was able to reach Day 15 with my last Xc-HUF-1 Differentiation protocol but was unable to get beating cardiomyocytes. Ran qPCR on the cells and saw that cTNT was expressed.
I am trying to generate iPSCs by retransfecting my target cells (HEK293T) grown on feeder cells. I grew Mit-C treated feeder cells (70% confluent) on Gelatin-coated plate followed by seeding HEK293T. I am transfecting the plate with my reprogramming factor containing plasmid after every 2 days. The growth medium is KO-DMEM + 20% SR + NEAA + L-glutamine + b-marceptoethanol. This medium is supplemented with small molecules like sodium butyrate, Ascorbic acid, LIF and rhFGF. I am changing medium after every 24 hours. Moreover, my plasmid is Dox-inducible so I am treating with DOX after every 12 hours. After induction, I can see the GFP signal from my transfected cells but no appearance of iPSCS. Instead of iPSCs, the cells are dying. I am using the amounts of all the above mentioned components according to the literature.
Is there anything wrong with my medium or strategy? Is it a good strategy to re-transfect my target cell line in a mixture of feeder + target? Are my cells sensitive to the applied cocktail of chemicals? Please share your expert opinions. It would be very helping if you share some optimized protocol. Thank you.
I've been attempting to expand an iPSC line but unfortunately, compared to other lines I've worked with, it grows very slowly. I nearly lost the line as it started to detach due to (I think) the matrigel giving up after 12 days. I managed to save it and get it to a nice stage with good colonies but to avoid detachment again I split the cells 1:1 onto fresh matrigel. However it is still very slow and I keep feeling I'm going back to square one.
I use StemMACs iPS Brew media and matrigel as per the recommendation of the provider of the line.
I'm relatively inexperienced with iPSC work so would appreciate any advice or suggestions!
In both our H1 and H9 cells (just bought/thawed) we have noticed that sometimes the colonies will die/lift after 2-3 days of passaging or thawing. Usually the whole well is affected, sometimes only the outer edges, other times only the center. We are not sure what is causing this.
They are both mycoplasma free.
We're pretty sure it's not our media, we make our own E8 but tested with Stemflex and mTeSR and we still see this.
Our matrix is laminin-E8 (0.1 ug/cm2), but we see it (but to a less exptreme) in geltrex (1:100) as well. Is it matrix related maybe?
Any help/input/advice woud be greatly appreciated!
I am trying to grow Embryonic stem cells and and I have read in the literature that rho inhibitor are used to preserve "stemness" and viability. These were the most commonly cited chemicals, but I have no experience with these pathways.
Hi has anyone had any issues using this kit? My negative control of the kit gives a higher value than the values suggested for positive control. Both controls from the kit itself. Tested for the recommended wavelengths of 490 and 650 nm. Also of course, all my derived mESCs show a high positive value. Suggestions/ Comments, please do share.
I used two cell lines hESC cutured in E8 medium feeder-free condition.When I convert into naive state use protocal by 3iL or NHSM media,I find the colonies shows continuous apoptosis and cannot passage even though forming a little dense colonies as picture.First I think the high concentration of every inhibitor harms to cells in consideration of individual cell line,but when I reduce the dose,it does not work.
Does anyone have similar experience or can give me advices?
Im currently culturing dental pulp stem cells but to get the media is kind of difficult. a colleague offers me alpha MEM instead of DMEM. can I change the media or the cells will die. I also have F-12 but i have heard that cells culture can fail. thanks for help or advise.
In terms of maintenance and differentiation, the potential of hESCs and mESCs differ. What are the differences between these two cell types and what are the ways to culture these two different cells? Please suggest a detailed protocol. Kindly cite any article if it is possible. Thank for your kind co-operation.
I have isolated tumor-initiating (cancer stem) cells from a solid tumor and have hypothesized that our molecule of interest mediates the differentiation of these cancer stem cells into neo-vascular tissue. I plan to incubate them in a hypoxic environment and measure the production of vascular markers and tube formation as described in the literature as markers for vasculogenesis with and without siRNA knockdown or inhibitor treatment
However, I can not find a clear idea about what media or factors I should add. There are many protocols for maintenance of these cells once they grow, but which media should I be growing them in? To force differentiation I see many researchers use VEGF, but A) I'm planning to measure VEGF production as an output for formation of these cells and B)I don't want to force them to become vascular cells, I want to see if they do it on their own.
If anyone has any direction or advice I would really appreciate it!
EDIT: I'm not starting with bone marrow or mesenchymal ES cells, these are de-differentiated astrocytes.
I want to culture Balb/c mouse bone marrow derived mesenchymal stem cells in Low glucose DMEM media but i am unable to culture them. Although when cultured in IMDM media these cells efficiently grow . Why I am not able to grow these cells in Low glucose DMEM media ?
Currently in the market for a protocol on how to culture hematopoietic stem cells isolated from mice OR if anyone has any suggestions on an immortal cell line representing immature myeloid cells. I am trying to study how cytokines change chemokine receptor expression in these cells.
I am working with neural stem cells from SVZ in mice. Usually I get these cells from 4-7 days neonatal mice. this time I got cells from 11 days neonatal and result of my cell culture is completely different. All the primary plates show just clumps of cell.
my media is DMEM(F12)+B27+ gentamycine+ glutamin.
Dose anyone know how I can treat these clumps and gets neurospheres?
We are using NS-A proliferation medium (Human) for neurosphere stem cell culture. Cell culture medium is becoming pink and little cloudy after every 5days during cell-proliferation. I am using sterilized condition all the time.
I was wondering if anyone can tell what could be the possible reason for this?
Thanks in advance ,
We are struggling with a huge problem since a long time in our cell culture. Although we culture the our iPS cell (E8 and its supplement with Geltrex Coating) according to the same protocol we always do, cells do not attach or come off day after thawing/passaging. We do not have any contamination, we have tried different incubators, different lot number of the cell media/coating/plates, different vial of cells (partially differentiated or pure iPSCs) and different cell culture rooms. Unfortunately we have got the same results (de-attaching or non attaching cells) each time.
Has anyone experienced that kind of problem before? If so could you give me some advice?
Thank you very much in advence
iPS cells were grown according to manufacturer's instructions:
E8 - vitronectin @ 400-500ng/cm2
mTeSR1 - matrigel (not specific concentration)
iPS cells in E8 apparently differentiate at a higher rate at similar conditions.
Room T-Culture media are changed every day.
Cells are split at 1:6
mTeSR1 seems to propagate and favor the growth of pluripotent cells while reducing the population of differentiated cells.
I am starting my post-doc using mESCs in a lab where they mostly culture human cells. They have a lot of human LIF from thermo fisher (PHC9481). I've read that mouse LIF receptor is activated by human LIF (but not viceversa), so I guess hLIF should be OK to grow mESCs in chemically defined media with 2i. Does anybody know for sure if this is OK? I am thinking on using 10 ng of hLIF / ml of medium.
I am using Mouse (ICR) Inactivated Embryonic Fibroblasts (Thermofisher, Cat No:A24903) to culture ES-E14TG2a Cell Line ( Sigma, Cat No: 08021401-1VL). It is suggested that approximately 1.5*10^6 MEF cells should be plated to culture mESC cells in T25 flask. If I increase density of MEF cells to 4*10^6 per T25 flask during culturing of mESC, Do I encounter any problem such as differentiation of mESC, low MEF depletion efficiency during MEF Depletion etc.?
Hello every body, I want to perform a fibroblast-like colony unit formation or CFU assay or clonogenic assay for mesenchymal stem cells.
Personally I am working with equine adipose-derived stem cells, I would like to know the optimal numbers to perform the assay in passage 3 because I am afraid of seeding too much and I cannot count them or too few and I do not see any colony. I thought about a range of serial dilutions (800,400,200,100,50 and maybe 25).
Another question I have, some people perform this assay with the stromal vascular fraction, which is better? Which number of cells I must use in this second case?
i want to culture neural stem cells from E14 mouse cortex. which neural stem cell culture gives the best result? what growth medium components are to be used?
for serum free media supplements? what do you recommend to use?
would using only GS21 be enough? or should i use b27 and N2 for better results? and also which vendor do you recommend to buy the supplements from?
thanks in advance
I'm dealing with human induced pluripotent stem cells culture using Matrigel or vitronectin XF coated plates. I passage the cells as small clusters and used ROCK inhibitor with mTeSR1 medium for the first two days of each passage. It has been fine for the past but recently, cells would detach and die. At first I through it was because they were too crowded and do the passage before it was too dense. It seemed to work. Then last time during passaging, I broke cell clusters into mostly single cells and supplemented with ROCKi all the way during culture. They grown normally for the first 5 days, then detach before reaching optimal confluency. I checked literatures but find nothing about long term ROCKi usage would cause detachment. Please help me find the reason.
My mouse embryonic stem cell line are not working. They are not able to attach to the surface of coated dishes ( I have tried both Geltrex and 0.5% gelatin) remain in suspension. I am using ESGRO LIF. Please help
Media composition = KO-DMEM+15% FBS+1xGluatmax+1xNEAA+BME+PS
I have successfully isolated stem cells from fresh urine but I am trying to figure out if the urine sample can be refrigerated for a period of time equivalent to shipping on ice and still get cells out. I am setting up an experiment to try and isolate cells from urine that is refrigerated for 24, 48 and 72 hours but I was wondering if people have done this. I haven't found many papers online. Any suggestions are welcome.
Is the only way to make essential 8 medium without FGF2, TGFbeta1 and insulin (I want to use insulin at 5ug/mL, not 19.4mg/L as in the original essential 8 medium protocol in the Chen et al 2011 publication) to make the medium from scratch? The commercially available essential 6 media (LifeTech) still contains insulin at 19.4mg/L if I'm not mistaken?
I am culturing stem cells for 96 hours and there are currently several Problems I am experiencing:
1- the humidity (even under the CO2/humidity/temperature control of cell culture incubator) does nto seem to be enough. Lecault et al. made an iso-osmothic bath, however, this is very time consuming and needs the whole design of the chip to be redone. Does anyone have other methods/tricks to Keep the environment humid enough for the cells in the chip? It seems the cell line cells surviving such environmental Change much better.
2- what are some surface covering techniques that have worked for you to reduce the pdms toxicity as well as reduce adsorbtion of small hydrophobic molecules into pdms? (the PDMS i have is 1:20 for control & 1:5 for flow layer)
thank you in advance for sharing any of your advices
i am new to microfluidics, so protocols, advices, tips and tricks on two-layer soft lithography are highly appreciated.¨
I am establishing a mo-DC protocol, and there is too many option in regards of maturation. I would like some help deciding. Since later in downstream applications I would be combining IFNg, I would like to include this cytokine in the protocol. However, I've seen that in order to truly create maDC i should activate the other canonical pathways. That is why I though of using TNFa and LPS. Before jumping and trying it out, I was wondering if anyone has experience with this type of cocktail or IFNg+TNFa.
Good evening, friends.
I did MSC differentiation from human iPSC. but difficult to maintain and subculture.
I followed the papers,
I just followed some published methods.
"PLOS ONE, January 2013 | Volume 8 | Issue 1 | e54524"
after 12 days, i subcultured on 0.1% gelatin coated dished and maintained in MSC media (Low glucose DMEM supplemented 10% FBS + rock inhibitor).
After 3 passaging, cells were not good.
After MSC differentiation, cell size was bigger than iPSC and numbers were decreased. there was no cell proliferation. I think cells are senescence. So it is hard to subculture and hard to maintain.
how to subculture iPSC-MSC? is there any media supplement or extra matrix?
Thanks in advance......
I want to culture spheroids and isolate stem cells from immortalized human proximal tubule cells. I have been able to get single suspension of human proximal tubule and seed them into ultra low attachment flasks but I want to pick one sphere and grow them on a 6-well plate to see if they can produce a clonal population. I am just beginning to do this, I do not have a lot of idea on these type of isolation techniques to obtain a stem cell population, so I would appreciate any comments/ suggestion. Thank you
i'm currently looking for what would be the best vector/plasmid for silencing genes in rat stem cells culture. I found some paper where they use lipofectamine, plus reagent, vector pCU and pCH. I also found that genscript make vectors. I would like to know f anyone does silencing with lipofecanie method, and also what vector would be perfect for silencing.
Thanks!!! and sorry for bad english haha
I am seeking a method to generate microglia from human mesenchymal stem cells (preferably bone marrow derived MSCs).
Does anyone have a method to suggest?
I recently tested the EdU alternative AmdU in MEF cells and tried to detect it using DBCO-Sulfo-Cy3 all from the company Jena Bioscience. However, the DBCO-Sulfo-Cy3 gave super strong unspecific staining everywhere in the cell. There are a few publications that used AmdU but different dyes for detection.
Can anybody provide helpful hints or a protocol to improve the staining?
Thank you very much,
Hello! I would like to ask about the quantification of sGAG after the chondrogenesis of mesenchymal stem cells.
My principal doubt is how to extract the sGAG to performs an assay with Dimethylmethylene Blue Assay (DMMB) because I have found protocols for that and they talk about the complex sGAG-DMMB but they do not mention how to obtain the sGAG in solution... Maybe with the application of the reagent the sGAG are realising to the medium as sGAG-DMMB complex directly after following the protocol?
Another issue, if I do Alcian blue/nuclear fast red stain. I cannot use them for the abovementioned assay right? I should have two pellets (one for the histology and one for the quantification)
Thanks a lot,
Hi, so I have hESC colonies growing on MEF feeder layer. I'm wondering approximately how many cells a colony of about 1mm in diameter would contain?
Thanks in advance.
I'm trying to do a live/dead cell assay (grown on 96 well plates) and I have problems with my cells adhering to the plate. I'm trying to establish a method that will allow addition of live/dead detection reagents, and then immediate fixation without washing out the live/dead reagents.
I need to fix the cells because this is for high throughput screening and I can't image quickly enough for accurate comparison.
I'm working on immobilize graphene oxide onto polydimethylsiloxane (PDMS) for cell line/ stem cell culture purpose. I have graphene oxide solution (not sure what the solvent is, but solvent is not highly volatile) and PDMS membrane/sheet. If I directly spin-coating or dip-coating graphene oxide solution on PDMS and dry it by heating, does it work to immobilize graphene oxide onto it? If it does work and durable for cell culture, I wonder the interaction force between the graphene oxide flakes and PDMS substrate. Or should I modify PDMS surface before graphene oxide coating? Any references?