Cell Culture Techniques - Science method

I am working with osteoblasts (hFOB cells from ATCC). After 5-6 subculturing their growth became so slow. It is about one week after the last passage and they are still not confluent. I have changed medium every 2 days. But, recently I realized that 2 days after changing medium, the medium become a little bit cloudy that makes it hard to see the cells. when I change the medium, it become more clear and I can observe the cells easily under the microscope. Do you think that my cells are contaminated? What can I do about this problem?

Thanks.

there is a protocol cryopreservation of BHK cells in 10% DMSO, and let incubating for 2 hours at 4 °C and then at -80?

I would like to label Human Serum Albumin for my experiments.

My supervisor suggested NHS and 6-Aminofluorescein as working agents.

Can anybody please suggest a working protocol?

Any suggestions on storing? Can I freeze the labelled protein in a solution?

If you have any other suggestions I would be very grateful!

I am having difficulties in reviving the chicken embryonic fibroblast cell line DF-1. I froze down the cells in complete DMEM medium plus 5% DMSO (half of a entire T75 flask) and thawed following the same procedures as other cell lines. However, even though the cells would attach on the next day, they started to die and float on day 2. I am thinking that the antibiotics (pen-strep) in the culture medium may affect DF-1 growth but shouldn't they only inhibit prokaryotic cell's protein synthesis (as for streptomycin)?? Has anyone come across the same problem?

I grown LNCaP cells in an RPMI media with 20% FBS. They are all tested and negative for any mycoplasma. They are being grown in a new incubator. It has been fully filled with water, 11 gallons on Thurs. Feb 20. The level of CO2 is 5% and was calibrated with a Fyrite calibrator on Wed. Mar 4. The temperature is set to 38C under ATCC recommendations and is calibrated with a thermometer that reads the same value. there is a medium sized water basin that is maintained at about half full and check every Friday. The cells present no signs of contamination, with the hood being in its own isolated room, opposite of the entrance into the lab, and is only ever accessed by myself, since I am in a two person lab. I sterilize all materials that enter the hood with 70% EtOH, wear a lab coat and gloves when in the hood room. I am not sure why the cells will not stretch and grow, but instead have decided to have a drop in the population.

Please let me know what I am doing wrong and what I could do to get these cells to grow. They were all growing great and working fine in the old incubator, but due to moving from one lab room to another, the new incubator has just been unable to reproduce the same conditions and are not allowing these cells to grow.

I have stored my LC540 cells in liquid nitrogen 3 months back and wen I try to retreive cells die. Can anyone help me out. S soon as I take vial out from LN2 I place them in 37 deg C waterbath and add complete medium to it and seed them. After 4 hrs I see cells attached from the next day my cells start dying.

Hi I've been trying to find a way to recover cells from matrigel for further culturing. So far the protocols I found are mainly for qPCR and use the cell recovery solution which needs to be kept at 4C for 1 hr to resolve the matrigel. However, I am trying to recover the cells and keep culturing them, so putting them at 4C for 1 hr would damage the cells. Is there a better way to do this? Can I just mix the matrigel with culture medium and pipet up and down to "dissolve" the matrigel? Should I then centrifuge the mixture and will the cells be at the bottom of the tube? Thanks! 

Hello everyone,

I am using hanging drop method to make 3D cell culture. But I am struggling with handle the spheres when I exchange the media.

Please help me if you have any experience with this problem. Thank you so much!!!

Best,

Usually people add M-CSF or L929 conditioned medium to regular medium (e.g. DMEM + 10% FBS) to differentiate bone marrow into BMDM (bone marrow derived macrophages) for 7 days.

I have an argument with my friends about the use of M-CSF (or L929 medium ) in the following cell treatments.

Do you use regular medium (DMEM or RPMI + 10% FBS) in the following BMDM treatments or regular medium plus M-CSF (or L929 conditioned medium)? We found the curves are different with different medium.

Thank you so much for any input.

I'm trying to grow PDX-derived cells from a SCCOHT model and I need them to survive short-term (7-14days) ex vivo

After tumor digestion I platted 7,000,000 cells in a 10cm ULA dish

In the following day they were forming spheroids but the spheroids were aggregating/clumping (First picture)

I filtered through a 40uM strainer and collect the portion that remained in the filter, washed with PBS, added 0.5mL of trypsin for 40s, neutralized with 1mL of 10% FBS media and them diluted in the serum reduced media (Counted with trypan blue and had >90% viability - Second picture)

I repeated this one more time after 3 days but they keep forming these giant aggregates every 2-3 days

I'm unsure if it is worse to separate them into single cells and lose the cell-cell contact or to let them grow in aggregates of spheroids

Does anyone know how to procced in this situation?

I digested the PDX tissue in Dispase/DNAse for 30min, filtered through a 100uM strainer, lysed the RBCs, minimized the debris with Ficoll and them platted in Advanced DMEM + 5% FBS

I've noticed most protocols use B27, only some use B27 minus retinoic acid.

Also, is N2 necessary, since B27 already contains components of N2?

Hi!

I want to make conditioned media from BMSCs in order to study their paracrine activity in other type of cells. I made the CM when BMSCs were at 90% confluence. Simply, I aspirated basal media (DMEM 1X, 15% FBS, 1% P/S, 1%NEAA, 1%L-Glu), washed 3 times with PBS and incubated the cells only with DMEM 1X for 24 hours at 37ºC. Next day, I collected supernatant, centrifuged it (to eliminate any cellular debris) and stored at -80ºC.

The question is... the cells which have been conditioned, can be used for do more aliquots of CM? I saw that in 24 hours many of them died, so I subcultured them in a shorter flask (from T75 to T25) and now, most of them look well but there are some that seem "damaged" as they were "stressed out" to make CM.

Looking forward to advices!

Many thanks,

Claudia.

Dear experts,

I would like to work on THP-1 as adherent cells without changing its natural. Is there any method or protocol for develop adherence cell from suspension cell lines?

I have an air-jacketed CO2 incubator and I need to calibrate its thermal conductivity CO2 sensor. However, I do not have a CO2 sensor that is independent of the incubator to measure the CO2 levels. Is there a method to calibrate the T/C CO2 sensor without having an independent instrument to verify its calibration?

Hi,

I have a simple question: When we freeze cells why we use 90% FBS and 10% DMSO?

I know that DMSO is used for cell cryopreservation but I don't know why we use FBS instead of culture media?

Has anyone performed stimulation of fibroblasts to myofibroblasts in cell lines. I found some articles on primary cultures. does the same work with cell lines?

Is it because we are trying to target these cells before they have a chance to divide??? I guess I don't understand the logic behind this.

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.

So recently I started working with HepG2 cell line and I'm having the hardest time to evaluate their confluency due to their morphology and the formation of aggregates, so I was wondering if anyone can tell me what's the confluency of the hepG2 in the photo and if I should sub-cultivate them in this state.

I have found a few questions here for this issue but I am not sure about the current admitted idea.

I want to make a drug treatment for establishing a resistant line and I will use a nucleoside analog. So, should I make my cell population synchronic before treatment? Of course, I will make cell viability and cytotoxicity tests on them.

Some researchers said the starvation or serum deprivation is not proper for making the cells synchronic, while some assumed this method is working. If I try this manner, how can I sure about its trustworthiness? And if it is working, how much durable is it? E.g; for the next passage after the synchronization, should I do it again and again?

Thank you!

My colleague and I are working with immortalized mammalian smooth muscle cells. We are re-using a modified 96-well plate. Thus far, this plate has shown no signs of contamination. However, to reuse the plate, we have to remove the cells that are currently growing in the wells. This was accomplished by aspirating out the old media, adding 0.1% (w/v) SDS to each of the wells, incubating the plate at 37 C for a bit, and then rigorously rinsing the wells with sterile water. Unfortunately, we neglected to filter-sterilize the 25% (w/v) SDS stock prior to making up the 0.1% SDS solution.

My question is this: What are the odds (qualitatively speaking) that some microbe--capable of surviving in 25% SDS--is going to destroy our cell culture? 

Intuitively, I think the odds are pretty low, since everything else was filter or autoclave sterilized, since we have not had any contamination up to this point, and since 25% SDS is a pretty inhospitable environment. 

But if I trusted my intuition alone, I would not be asking this question, right? I am new to this field. Have other people ever encountered this situation before? Has it led to problems?

Thank you for any advice you can provide!

Hi Guys

For the last couple of months my lab has been riddled with infections. All cell lines. It is odd though in that the infection is only visible the day after transfection with either CaP or Lipofectamine 2000. These are not typical bacteria – they are absolutely tiny and don’t swim, but appear to wriggle. It is not Brownian motion and is definitely infection of some sort as confirmed by my confocal microscopy staining.

My working theory is that the bacteria (I feel it is mycoplasma…) are INSIDE the cells until we “stress” the cells by transfecting them, whereby the cells burst open and release the bacteria into the media. I have not seen this online so I am not sure. Has anyone seen this before?

I am aware you cannot really see mycoplasma under the light microscope but the “wriggling” things could be colonies, and my confocal appears to indicate mycoplasma infection.

We have absolutely no idea what else it could be at this stage as we have cleaned everything four times, filtered all reagents…. Everything!

Does anybody have any suggestions as to how to solve this issue? We are currently treating some cell lines with Plasmocin and any cells we “hope” are not infected are being cultured with a low dose to prevent mycoplasma infection.

It is absolutely insane how this infection won’t pass! I think it must be mycoplasma as I have started using both Gentamycin, Amp B AND Pen/Strep in my media to ensure/kill bacteria. And yet… we are always getting infections BUT only apparent following transfection. It is not the transfection reagent as I have used different brands, batches and even tested all individual reagents on cells (ie. Added a few microliters of DNA, Calcium Chloride, HBS, PBS, FBS etc…) and no infection is seen. It seems stress induces the exit of the bacteria from cells.

Has anyone seen this before?

Please help if you can!

We work with OKG4 gingival Keratinocytes between Passage 13-18 and cultivating them on Collagen 4 in 75cm Flasks and implement them with 2x10*4 cells on 96 Well with 0,6cm growth area and 5x10*5 cells in the flask again. Both products are from Falcon and coated with same concentration of Collagen 4.

There are immense differences in growth and morphology while both products were treated in the same way. The flask show much better results, while the cells in the wells could only be saved after several washings and medium exchanges.

Our Collegues work with HeLa- cells in the same incubator.

We are open for Helps in each directions!

I wonder if anybody used Lipogro or Fetalgro for cell culture? Does either match or outperform FBS for cell lines?

Hi - I am going to isolate some exosomes from cell culture media soon (with cells grown in exosomes depleted FBS for 24 hours).

However I would like a positive control on the isolation and was thinking about using non-exosomes depleted FBS.

My question: Does anyone know how many bovine exosomes are present in "normal" FBS per ml?

Thanks in advance,

Gary

I am new in cell culture techniques, I am maintaing 231mdamb cell lines right now, cells seems healthy and growing well, but I observe in one flask cells are growing in rows inbetween scatterted cells, I am attaching pic.

These questions may sound silly, but this is all quite new to me! I have two queries:

1. If I grow my cells in 24/6-well plates, is it safe to trypsinize the cells first then lyse in buffer? Or should I scrape cells directly off the plate, then lyse?

2. Since the kits I purchased didn't come with lysis buffer- can anybody refer me to a formulation of this?

Hi all,

I am an undergrad in a physiology lab. I am investigated the role of ferulic acid on certain markers. My PI proposed an experiment involving the overnight loading of high ferulic acid esterase producing bacteria onto cell culture. I'm having trouble finding a similar type of protocol in literature. I was thinking of heat-killing the bacteria, since FA is not a protein so potential aggregation would not be a problem. I'm unsure if there is a better technique available and I am simply lacking the term for the technique to looking into. Any help would be greatly appreciated.

Thanks

Romina

I have been doing cell culture with B50 cell lines recently and the cells have not been showing the expected neuronal morphology, even after reducing the serum concentration. I just happened to do a check of the osmolarity of my cell culture solution and turned out to be 407 mOsmol/kg, which seems to be quite high compared to literature values.

My DMEM contains: 2.5%FBS, 2mM Glutamine, 10uPenstrep, Na-bicarbonate (3.7g/L), Sodium Pyruvate and HEPES.

I am adding too many things in the plain DMEM? Does anyone have an idea of what adverse effects this high osmolarity might have on neuronal cells? Could this be the reason for the bad cell morphology?

Will be thankful to any insights on this!!

Bacterial pellets seems to dissolve readily when put directly in LB-media is it necessary to put them in phosphate buffer first?

I'm preparing a micronucleus slide but I see my slide is very dirty. I'm using normal lymphocyte with carnoy fixative and I'm drying it in slide with air and 10% gimsa stain. How do you prepare good and clear micronucleus slides with high quality?

What is the recommended seeding density for H9C2 cells (specifically in a plastic tc t25 flask)? We have been seeding at the recommended 10x10^4/cm^2 (250,000 in t25) and they are reaching ~70% at 48 h instead of 72 h. The majority of our cells seem to be attaching. Can we seed at lower amounts without changing the cells? Additionally, how many cells would be in a confluent t25? We are using the media and trypsin specified by ATCC.  

I have gone through quite a few papers, and protocols. .Majority of the papers mention the addition of IL-2 to maintain Tregs in-vitro, however, some papers use both IL-2 and anti CD3. Which is the preferred methodology and why?

Thanks

I was wondering if there were any alternatives to using FBS/FCS in the Min6 cell culture media.

I am treating RAW 264.7 macrophages with some plant extracts dissolved in ethanol and measure if they decrease ROS/NO production. I have been having some trouble measuring ROS.

A condensed protocol is as follows;

1) Seed 500K cells, in 500ul volume per well in a 24 well plate 37C and 5% CO2. Let them adhere for 3-4 hours.

2) Add 2.5 ul of H2DCFDA 5mg/mL per well

3) Incubate for 30 minute 37C and 5% CO2.

4) Aspirate media, wash cells twice with warm PBS.

5) Add 500ul DMEM media.

6) Add 1ul of plant extract with [50mg/mL]

7) Incubate for 1 hour at 37C and 5% CO2.

8) Add 10ul of LPS [100ug/mL]

9) Incubate over night (18-20 hours) 37C and 5% CO2.

10) Read fluorescence in that same plate at 485nm excitation and 520nm emission in a plate reader.

Somehow the numbers are all over the place. Can anyone suggest improvements? How do you measure ROS in your lab? Should I change the incubation time?

Any feedback is appreciated and I would be more than happy to clarify/expand on the protocol details,

Hi everybody. Does somebody know a reliable human osteoclastic cell line? I'm searching a good model, but I find almost only murine cell lines.

Thank you,

Cristina

I have been conducting an experiment on HeLa cells; I do not need phosphate contamination or at least minimize phosphate interaction with my culture as much as I can. Thanks

Recently I have been problem with HEK293a expansion problem, after 2 passages in flask, the cells stop growing and detach from flask. I am cultivating using DMEM 10 % FBS (High glucose, 10 mM HEPES, 0,2% sodium bicarbonate, 59 mg/L penicillin and 133 mg/L streptomycin) all from Sigma. I need to expand this cells to 16 flask of T-150 (Corning). I don't know what is wrong. I would love to hear with someone had a similar problem.

I wanna get NK cells from PBMC (CD3+ cells will be depleted by magnetic cell isolation MACS for culture), but most protocol require irradiation once every 2~3 days during PBMC culture(~14d).

but the problem is we don't have irradiator. So I want to find the way I can get NK cells without irradiation step.

How can I get NK cells from PBMC without irradiation?

We are trying to isolate keratinocytes from skin using a house made selective keratinocytes culture medium and fibroblast as feeder cells. We have already seen keratinocyte colonies and we have expanded them using our house made culture medium. 

We are used to work with HaCaT cells and we use low calcium DMEM to maintain them in cell culture. Do we have to culture primary keratinocytes like HaCaT cells, or do we have to maintain them in their selective isolation medium?

Thanks!

ATCC website says CHO-K1 cells require proline in their growth medium.

We have been culturing CHO-K1 cells in our lab for many years in DMEM+FBS. DMEM does not contain proline. So how are the cells able to grow? Are they getting proline from FBS?

Good afternoon,

I have some question related to the handling method for neuroblastoma cells SH-SY5Y.

First, I grew the cells with EMEM medium (10% FBS and 1% Penicillin/Streptomycin). The cells grew well and started to divide. However, the cells aggregated and formed clumps, and the 48H later most of the cells detached from the bottom of the plate and floated in the medium. Since the cells did not attach to the plate, we found it hard to handle and do experiment with the cells.

Therefore, we tried another medium, which is DMEM( 1,000 mg/L- Glutamine, High glucose 4.5g/L, 110 mg/L sodium pyruvate) + 10% FBS and 1% Penicillin/Streptomycin. The cells also grew well at first, but we faced another new problem. The cells did not form many clumps like the first method, but the shape of the cells were changed and some of them still detached from the plate and floated.

(Note, I am aware that there are 2 forms of this cells- adherent and floating forms. But i was told that the floating one is not good to be used in experiment and also I need the attached cells for my experiments)

Hence, I am looking for your recommendations here. Any opinions from all of you are highly appreciated! Thank you!

I'm using primary cells of keratinocytes, but I have problems in subculturing them. They grow well during passage 0. But after subculturing most of them are gone; in other words they did not attach to flask. Dissociating reagent was TrypLE Express and the culture medium used was CnT 07. Anyone have solution? Thanks in advance.

Some people suggested to use TSB with blood or bolton broth for reviving and 20% glycerol for storage. As this organism is fastidious, could someone who has the experience of working with Campylobacter  please explain the better procedure. 

I'm curious whether anyone on here has experience with InvivoGen's PlasmoTest to detect Mycoplasma infection of cell culture lines, as well as perhaps how it compares with PCR and Hoechst staining.

I know the gold standard is direct culture, but at best we would just outsource that to a commercial service.

Hello all,

I am performing a kill curve assay for the first time and need some help with these simple dilution calculations.

One for example, I have a stock antibody that is 50mg/mL. I need to make dilutions of 800ug/mL, 600ug/mL, and so on. I am plating the cells in 200ul volume in a 96-well plate.

Can someone walk me through the rationale for the math? How much antibiotic do I use to well with 200ul media/antibiotic?

I am using NEB PCR cloning Kit. There is pMiniT vector inside kit. I transformed ligation bwt my PCR product and pMiniT vector to NEB 10-beta competent E.coli cells. I used SOC growth medium for transformation to increase efficiency. I got colony on LB-agar plate. I inoculated some of these colonies in LB medium. However, none of my colonies didnt grow. Should I use different growth medium?

I need to prepare some M9 minimal media but previously the solution has become cloudy after preparation.

Can anybody suggest what I am doing wrong?

Thank you,

Jack.

I am incubating cells in RPMI in a CO2 incubator at 37C and 80% RH. The phenol red started as a pink color and after 24 hours in the incubator it has faded. The color is more or less the same, just less bright. Is that normal?

Hi,

Our B16F10 cell line from ATCC encountered massive cell death after switching the growth media from RPMI to DMEM.

We were stuck with RPMI( 10% FBS, 5ml P/S) for the first 2 passages with the lab mistakenly ordered DMEM w/o sodium pyruvate & l-glutamine. The B16F10 cells looked healthy as we split them at the subcultivation rate of 1:5 and passaged them every 3-4 days.

Then, the growth media was replaced with DMEM when the glutiMAX arrived. 5mL of glutamax (5mM) + 10% FBS, 5mL p/s was added to prepare the DMEM. The cells were split 4 days ago with the new DMEM and at a much lower ratio of 1:12. We checked the cells today and they were all dead. Shown in the pictures.

Any advice for the troubleshoot would be very much appreciated! Thank you in advice for the help!

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.

Dear all,

Hello! I have had issues with Hek293T cells maintenance for quite awhile whereby my Hek cells die after passages 6 or 7. I've recently started trouble shooting to find out the source of contamination.

I discovered white spots in my co2 incubator as well as white fuzzy substances in the water bath tray of the incubator. Are these signs of possible fungal contamination? I've also inoculated a 12 well plate with DMEM (only), DMEM (only) + FBS, DMEM (used for Hek293T cells maintenance) + FBS and DMEM (used for Hek293T cells maintenance) [ALL WITHOUT ANY HEK293T CELLS!!!] and incubated in the potentially contaminated incubator to determine wether the contamination was due to the incubator or the media/serum we've been using in the lab. Upon looking under the microscope, I saw thread/string like structures. I've checked on it a couple of days later and saw more of these structures under the microscope as well as some other structures that I am not able to recognise. Are those resulting from a potential fungal contamination?

Please help! I've attached some images I've took as well!!

Hi everyone,

I'm having cell growth problem in outer wells of 96 well plates for bioactivity assay, recently.

The problem occurs during the incubation in the 37oC CO2 incubator. While 96 well plate is treated with the cells, firstly 200 ul PBS is added into the outer wells of 96-well plate as shown in the attached figure (wells colored in grey). Afterwards, cells is seeded into each well of the 96-well plate so that each sample can be treated in triplicates, as shown in the attached figure (wells colored in white) then plate is treated with the standard/test solution. After the treatment plate is incubated in the 37oC CO2 incubator for 72 hrs. By the way, I have used these seeding protocol other times, so I know they work.

During this incubation, no growth is observed in the outer wells containing cells (e.g. B2, B3,C2, D2, E2, F2, G2.). To overcome this problem, PBS is added to the gap between two wells too. Still, the problem was not completely solved. I think the problem is caused by evaporation in the incubator. If you may have an opinion about the reason of this?

I look forward to hearing from you.

Thanks in advance :)

I am using cell culture supernatants in an ELISA. The cells are incubated at 37C, centrifuged, then the supernatants are used in an ELISA at RT. Why does the supernatant collection need to be at 4C if all other steps are at RT or warmer?

My MNFS60 cells are dying suddenly after overnight incubation . I propagate cells in RPMI 1640 + 10% FBS + 1% Penstrep + 0.05 mM 2-Mercaptoethanol + 62 ng/ml MCSF as recommended by the cell line provider. I have tried varying different batch of media, different incubators, vessels, ordered different MNFS60 batch etc. All the chemicals are newly ordered. Mercaptoethanol dilutions are prepared fresh. My incubators were validated and checked for temperature and CO2 just 2 days before the cell death. Materials are stored at recommended temperature. Important point that i noticed was: the alliquote of cells used for cell count was left at room temperature by mistake. The cells in it were viable next day. It did not contain mercaptoethanol and MCSF. Where as the cells that I incubated the same day, died!! My incubators always show a slight colour change in the media (orangish yellow) with and without cells.

We have some pre-made RPMI from 2014. The manufacturer says it lasts a year in a fridge but I think that is just a ruse to get us to buy more. It was never opened. Could it still be good? Can I just compensate by using more?

- When I performed ICC work for treatment cells in translocation stress experiment in IBIDI 12 well removable chamber I got variant signal intensities ...

What is the reason behind cells stress without any kind of treatment!

Hi!

I an interested in culturing cervical kerationocytes and calcium levels in media are know to affect their cell growth. I recently obtained two KSFM for the same (one with Calcium and another without). I havent been able to get the exact calcium concentration for the 1x ksfm cat number-17005042 and would be happy if someone can help me with that. Also, what would be the ideal calcium concentration be that would suit keratinocyte growth?

Many Thanks.

Swetha

I have the XTT salt (powder) and I would like to use it for XTT assay.

For that reason, I am going to follow the Roche CellProliferation Kit which constitues of:

- XTT in RPMI 1640 medium,1 mg/ml, filtered through 0.2 μm pore size membrane (I am going to use DMEM instead of RPMI since I don't have RPMI in my lab right now)

- PMS (N-methyl dibenzopyrazine methyl sulfate) 0.383 mg/ml (1.25 mM) in PBS, filtered through 0.2 μm pore size membrane

The kit is meant to be stored at at -15 to - 25°C.

I plan to prepare the XTT and PMS solution as described. Do you think it's ok to prepare more of them than just for a single use and to freeze the solutions? The XTT solution contains cell medium which raises my doubts. Is it possible that the manufacturer of the Roche Proliferation Kit puts something more in the solutions (cryopreservative?) that is not mentioned in the composition?

I attach the Roche Proliferation Kit leaflet file.

I would be grateful for any responses. Thank you.

I grow LC-540 Leydig cells, the problem with me is as shown in the picture a. The ones rounded in yellow circles are black particles attached to the plate loosely it does not show any movement, but once i wash those cells with PBS it gets removed off easily as shown in picture b. After subculture i get back these particles.

But i feel like these are dead cell particles and i am not sure if this is contamination too..

Please help me identify the problem and need suggestion to get rid of these.

Hi All,

I would like to check whether commercially available cryovials with silicone gasket, internal thread are safe enough to store in liquid phase of nitrogen tank or not. I see all labs are practicing their own way. They said no problem and can dip into it. Some said no, the liquid can go into no matter with silicone seal or inner or outer thread and suggested to store in vapour stage instead. (above the liquid). Available cryoflex is not also convenient to use as it may take time to do it and still exist tiny spaces for liquid to come in too. Very confusing. What is the best method to seal the vial before putting into the cryobox of LNT. Are there any special seal? How about wax? Aluminium tape? or Silicone paste to wrap. Paper tape?  

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!

I work on evaluate the cytotoxicity of herbal extract on primary human monocyte-derived macrophages (MDMs). When I'm doing MTT assay on 96-well plate, I scrape MDMs from cell culture flask and seed in 96-well plate with density 50,000 cells/well. However, after overnight incubation most of MDMs are not attach to the surface and lost during washing step in MTT protocol. So, I think it might come from the high density of the cells and there is no sufficient surface area for MDMs to attach(?) or the cells just died after cell scraping (?) Could anyone please suggest me on the optimal density of primary MDMs in cell culture plate or other tips for handling with MDMs culture would be greatly appreciated.

The yellowing of the media indicates the cells have an increased metabolic rate. The yellowing only occurs with the DM cocktail; when the DM cocktail is replaced with maintenance media (DMEM + 167nM insulin) on Day 2 the media is still healthy on Day 4,

At the end of the differentiation program (14 days after induction), I only get small patches of differentiated cells; the vast majority of the well contains undifferentiated cells (I run the experiment in a 6-well plate). The cells have been strictly sub-cultured and to my knowledge, are relatively low passage. 

Here is a run down of the protocol I use: preadipocytes are grown to 2 days post-confluence in DMEM supplemented with 10% FBS +1 x P/S (day 0) and the medium changed to DMEM supplemented with 10% FBS, insulin (167 nM), dexamethasone (0.5 μM), isobutylmethylxanthine (IBMX) (0.5 mM) and rosiglitazone (2μM). After 48h, the medium is replaced with medium containing DMEM supplemented with 10% (v/v) FBS and 167 nM insulin. On day 4, after inducing differentiation, and thereafter, the cells are cultured in DMEM with 10% FBS. This maintenance medium is changed every 48 h until the cells are utilized for experimentation.

Thiotone E Peptone (ref 212302) has been discontinued. Could you recommend an alternative product?

Thank you

As part of my Bachelor Thesis, I'm working on a protocol for expression of Caspase-3 in E. Coli (and subsequent purification). I heard once that the protein yield can be increased by not only having a preculture but even a preculture of the preculture itself (Hence, pre-preculture). Does someone know more about this topic or recommend literature?

Hello everyone!!

I am trying to grow MIN6 cells, but I am unable to. The growth rate is too slow. I can hardly see 5-6 cells in the plate. Also the morphology of the cells is highly irregular. I am using DMEM plus 12 percent FBS with high glucose. Any suggestions are highly welcomed.

TIA

Dear All,

What is the best way of picking the positive clones while establishing a monoclonal cell line expressing your desired transgene. I am currently doing it with a P-20 pipette under a light microscope. However, it is very hard to tell whether I have specifically picked the single clone or not. Is there a better way.

Thank you

Ikram

Hi Everybody! I was culturing HL-1 cells in Claycomb medium with all needed supplements and they were growing nicely but I ran out of that medium and tried to culture them in DMEM. The cells were continuing to grow but they were also dying in great numbers. I wanted to ask if anybody figured out what is ''the secret portion'' which I could supplement to DMEM so my cells could live happily ever after ? :)

P.S. Claycomb medium is very expensive but nevertheless I ordered it. And I have to wait at least two months to get it.

This concerns linear PEI 25kDa. So far I am aware of two methods:

1: stir the PEI in MQ (1mg/ml) and dissolve by dropping and holding the pH at 2. After its transparant  increase pH to 7 --> filter through 0.22um filter --> aliquot --> 4x freeze-thaw.

2: stir PEI in MQ (0.323mg/ml) and dissolve by heating to 55C O/N. Next day, the solution is cloudy --> cool down to RT --> adjust pH to 7 (still cloudy) --> stir again at 55C and repeat this until its not cloudy anymore and pH is stable at 7. Then filter through 0.22um filter --> aliquot --> 4x freeze-thaw

I'm trying both. #1 works is fast and works well. #2 takes a lot of time before the solution is transparant. 

Does PEI dissolve completely? And what is the best method to dissolve PEI to use for in vitro transfections?

I am trying to improve the survivability of primary neuron cell cultures. Currently, I am getting great survival out to 20-days but would like to push my time point out to 42 days. I currently believe that the neurons are being crowded out by microglia so I am looking for ways to reduce their growth. Additionally I am curious if anyone has had any experience with 3D cell culture techniques and could provide some technical expertise and thoughts on this method compared to standard 2D cell culturing.

Hello everyone,

I am currently trying to set up at a new lab in order to begin experiments on osteoclasts. Before starting anything, I wanted to make sure that BMMs have indeed differentiated into osteoclasts by using TRAP staining and PCR.

In our lab, we harvest bone marrow-derived macrophages (BMM) from the tibia of 5 week-old female ICR mice for osteoclast differentiation. For BMM differentiation, I seeded 2 x 10^5 cells per well with full alpha MEM and M-CSF (30ng/ml). I used 3 6-well plates in order to retrieve cells on Day0, Day2, and Day4 of differentiation. On the next day, I retrieved the Day0 plate using 1ml of Tri-RNA reagent and changed the media (containing M-CSF and RANKLE (1:1000)) for the other two plates. I retrieved the rest of the plates on appropriate days.

After retrieving all cells, I performed RNA isolation followed by RNA quantification (ND-1000), reverse transcription, PCR, and gel electrophoresis. My problem here is that I'm getting nothing on gel for Day0 with actin, GAPDH, and HPRT primers. I triple-checked all my steps for gel, PCR, and reverse transcription using other cells and the technique does not seem to be the problem. I performed RNA and DNA quantification using Nd-1000 (I know they are not super accurate) and I've attached the results as image files.

Please help me figure out what made the Day0 bands disappear! Thank you in advance:)

I am trying to find a media recipe for the MCF10A cells. I have ordered the MEGM kit from lonza that comes with MEBM media + bullet kit of growth factors. The protocol from brugge's lab gives exact quantities of the growth factors to be added which is different than the quantities provided by lonza in the kit. Should I add the same amount mentioned in that protocol or the entire vial content given by lonza?(vial amount of EGF and hydrocortisone is higher that the mentioned amount by Brugge's lab protocol). will it harm the cells if i add less?

I'm interested in using Sircol Collagen Assay kit to determine if my stimulated renal cells are inducing collagen synthesis, does anyone have experience with this? i'm using adherent cells in culture, thank you!

Hypothetically, could someone survive by consuming DMEM with L-glutamine? I understand this is not recommended. I just wonder, because DMEM is formulated for human cells.

Thank you.

Hello,

I'm tryting to perform an adhesion assay adding THP-1 labeled with calcein to a monolayer of endothelial cells previously stimulated with LPS or TNF-alpha. However, I realised that THP-1 labeled tend to attach to the bottom of the culture plate (which they don't do normally because they are suspension cells), so I have a problem with unspecifity adhesion and I don't consider my results reliable. Have someone perform this assay and have some solution or "trick"? I do it in 96-well culture cell plates.

Thank you.

Hi,

we will be soon working with CHO-S cells for recombinant proteins expression. We are deciding which media is the optimal to work with. Our first choice is FreeStlye (LifeTechnologies) for growth and CDOpti CHO (LifeTechnologies) for maintenance after transfection.

A colleague (that worked with these cells and another protein like 4 years ago) told me that she tested Forti CHO (LifeTechnologies) (with different supplement combinations) and ExCell CHO (Sigma) and she wasn't unable to notice any difference in protein production.

Do any of you have any media recommendations we should take note of for better protein yields?

Thank you very much.