Cell Culture - Science topic

Dear all,

I found in cell cultures some structures that I cannot identify

I will really appreciate is someone would give me some suggestions

Many thanks

Francesco

Hi everyone

I produced a batch of virus , enriched it with centrifugation, and titrated it with T cells after 48h. For my experiments I want to use MOI 3 and that's equal to 1/12 dilution.

but at this dilution my cells will die after 5 days in cell culture( I use PBMC not sorted T cells), while untraduced control expanded 6 times during 5 days of stimulation. Until day 3 transduced cells are similar as untraduced cells. I tried up to 1/160 dilution but I still have very large cell death with this dilution!

Do you have any idea what is the reason for this?

Thanks

Hello all. I grew some cells for virus infection. I infected the cells with CMV (Cytomegalovirus) 8 days ago. The cells have not started lysis yet, but the plate is about to get dry. I wonder if I can add 5 mL of growth medium (DMEM) to the plate containing CMV-infected cells to prevent dryness at this moment (8 days after infection). Is there any risk to do so? Any advice is appreciated.

Hello all. I grew ARPE-19 cells in cell culture and infected them with a virus (Varicella-zoster virus). After the virus reached a high infection rate, I harvested everything in the plate and use freeze & thaw technique to release viruses into the supernatant. Now I want to store my viruses in -80 freezer. What is the composition of freezing medium for VZV? Are DMSO and FBS enough? Or do I need to add sucrose or something else? This is a bit urgent. I have no one to ask because I am the last employee in my lab as my professor is retiring. Any advice is appreciated.

I observed this clumps in my MCF-7 cell culture. Does anyone know if it is normal or contamination?

Hello, I have isolated extracellular vesicles from a cell culture using the Exosome isolation kit. However, the western blot results did not show up because the concentration was only 0.2 ug/ul. I believe a concentration of 1 ug/ul is required for a successful western blot. Could you please suggest a method to increase the concentration?

Hello. I am new to cell culture work. May I know whether these small black dots are contaminants or just cell debris? It's quite confusing because they are not moving. This is different from the contamination issue that I used to encounter because I can surely confirm that my cells are contaminated when these small dots move which means bacteria. FYI, this is HEK293T cells.

Just wondering if anyone has any experience in setting up a Getinge mini bioreactor? More specifically, autoclaving the reactor, the gel pH sensor, and the dO2 sensor.

I am having trouble understanding how to use the pH probe and "pressurize" in the autoclave before first use. The pH probe has a lifecycle of 10-15 autoclaves. Would I have to bathe the probe in ethanol as a method of sterilizing the probe between cell cultures?

Any advice is welcomed! Thanks so much in advance!

Greetings,

My question is what insulin concentrations would be used to examine the effect of hyperinsulinemia while my growth media already supplemented with 5ug/mL insulin to support cell culture?

And;

Thanks,

Maryam

Hi all, I have a set of cell culture samples embedded in 1% agarose. They have been cultured for 14 days. They have been fixed with 4% PFA. I am wondering if I can separate the whole cells from the agarose gel from these fixed samples and test proteomics from both the gels AND cells separately?

Hello dear researchers,i have transduced my HEK293T cells with IL-2 and then puromycin screening was done, the cells were showing good egfp flourescence ,but then due to some reasons I have to store my cell culture at -80 degrees,and now after 2 months if I thaw and regrow my cells, will they be able to produce sufficient interleukin 2 protein?i am a little worried...

HI

i transfected adenovector to hek293 cell line. i don't know Adenovirus CPE.

this picture is hek 293 cell line 8 days post transfection. do you observe any CPE?

Because so far, the petri dish we got are only treated at the bottom part of the petri dish not the lid. Can the spheroid still form?

I'm a current medicine student and I'm in my first scientific initiation in my graduation. Currently, me and my professor are having some troubles with contamination in our cells. This is a bottle with HCT-116 cells (colorretal cancer). This cells were, previously frozen with contaminated fetal bovine serum. My professor said she thought she treated the cells, but this contamination keep returning. We supplement our media with penicilin and B- anfotericin at 1% each. The weird thing is: The bottle keeps healthy and, suddenly, from one day to another, these clouds appears and the bottle liquid looks cloudy. Could anyone, please, help me by advising what could I do or what fungus is this (If this is a fungus)?

Hello everyone,

I am facing a frustrating issue with the TC28a2 cell line. After treatment with staurosporine at concentrations of 25nM, 50nM, and 100nM, the cells begin detaching during the first media aspiration when I switch to fresh complete media. This problem occurs regardless of the incubation time. Does anyone have any advice on how to aspirate the media without losing cells?

I am encountering a challenge with culturing endothelial cells derived from umbilical cord tissue. Despite successful isolation, the cells exhibit a decline in viability and begin to deteriorate after 3-4 days in culture. I have attempted to address this issue by utilizing various brands of endothelial cell media, yet the problem persists. Enclosed is image depicting the condition of the cell culture flask. Any insights or recommendations regarding this issue would be greatly valued.

Hi,

I have been cultivating Jurkat cells in RPMI supplemented with 10% FBS, and I have observed some small things in my flask. Although the cell growth appears unaffected, I want to ensure that this isn't contamination.

Can you help me?

For H202 radical scavenging assay. How to prepare 75 uM hydrogen peroxide from 30% H2O2 solution ? Thanks in advance.

I am studying the anti-tumor effect of Hoechst33342 dye on A-375 cells. However, when I added 75uM of the dye to the plated cells, the dye seem to precipitate and I get close to 100% cell death. Is this normal? If not what can I do to avoid this precipitation? (the stock Hoechst33342 solution is prepared in distilled autoclave water)

We are working on a prototype cartridge for cell culture applications and I need to adhere a polystyrene well plate to a polystyrene coated glass micro chip. Looking for double sided adhesive tapes that are biocompatible and cell culture compatible. Preferably with resistance to ethylene oxide sterilization. Any suggestions?

Dear Researchers,

I have a couple of falcons with collected media from cell cultures. I have been storing them for a year in 4*C. They were collected for an experiment that I hadn't had the chance to finish. My question is, can I perform any analysis on them while they were stored in such conditions for this long? Anything with DNA/RNA/proteins? Or everything is degraded by now? I will appreciate every idea.

I cultured Human Umbilical Vein Endothelial Cells (HUVECs) on a plate coated with collagen type 1 (rat tail) at a concentration of 7.5ug/cm^2. The coating procedure involved dilution with cell culture water, incubation for 1 hour at 37°C, followed by removing the solution and washing twice with PBS. Subsequently, I used EGM-2 medium for culturing the cells. Everything appeared normal on the first day, but by the third day, the media became cloudy, and most of the cells were either detached or not visible. Please refer to the attached video for more details. Any insights or suggestions would be greatly appreciated.

I'm just wondering if I need to change the media of my cells that are culturing in 5% O2 (hypoxic condition) in a hypoxic chamber aka a hypoxic glove box? Or can I just quickly do this in a normal incubator and return the cells to the hypoxic incubator?

It sounds like a lot of work if we need to get a hypoxic glove box so I'm just wondering if anyone has any experience in this who could give me some insight?

Thank you

Dear all,

I prepared my cell media (EMEM+10%FBS) and another cell media (EMEM+2%FBS+1% Pen/Strep). Incubate the aliquot of the media in 37°C for a few days and saw these. They are not moving and there was no color change in the media. Since there were no cells in the flask so if these weren't cell debris, I wonder if this is debris/aggregates from the serum? I also have done sterility check on the serum and no changes observed to indicate contamination.

Thanks.

I'm expading MSC (Mesenchymal Stem Cell) from umbilical cord uner 37 °C with 5% CO2 in a humidified chamber.  I'm testing 2 types of media, both appropriate for MSC (StemPro and NutriStem).  I need to expand the cells, but they keep differentiating. Does anyone know what might be happening?

Hi everyone,

I have been having some issues with SHSY5Y cells that I never had in the past. I used to culture them in DMEM:F12 with 10% FBS and 1%Antibiotic-Antimycotic and they grew just fine. Back in October, when we received a new batch of medium, I started seeing increased cell death after 1-2 passages: specifically, I would see debris like structures and the cells literally peeling off from the bottom of the flask. I initially attributed this to the quality of the medium and ruled out incubator, FBS etc issues. I thawed a new vial of cells and I had the same issue. Then I was told to try OptiMEM and with a new vial from ATCC I noticed that they grew beautifully and reached confluence within 3 days. After the 3rd passage, I started seeing the same type of pattern that I saw before--cells fragmenting, a lot of debris and cells peeling off and floating in suspension 1-2 days after plating. I am desperate as my lab has wasted many cell stocks and money on trying to figure out what this is. My next step is to test for mycoplasma but I wasn't sure if this is the case considering that we literally just bought a new stock from ATCC and it grew fine until the third passage.The medium doesn't yellow out and it is not yeast contamination. What can be the source of this type of behavior?Any input is greatly appreciated! :)

Hi all,

I am doing a transient transfection to my cells with GFP-tagged plasmid and sending them to be FACS sorted and I need to replate them once I get them back. This is my first time ever doing this kind of experiments. would you please kindly help on how I could replate them? is it casual cell culturing? I heard that you send your plate and get a tube back?! would you be so kind to elaborate? Thank you

Hi everyone, I am trying to culture IL-2 independent NK-92 cell line for weeks now but they are still not proliferating.

Media used: MEM with 10% NU serum, 0.2 mM myo-inositol, 0.02 mM folic acid, 0.1 mM BME, 1 % Penstrep

After centrifugation, I resuspend the cells in 10 mL of media and placed in T25 flask. Every other day there is still no sign of proliferation nor high density clumping (Almost 3 weeks in culture now). All I see are crumpled cells but in a clump manner (I assume these are dead cells). This is my first time culturing NK92 cells and I'm still not familiar with how they should look like and troubleshooting them. Thanks in advance for any inputs you can provide .

The Advanced reduced serum DMEM/F12 works fine for my cell cultures, even without adding any serum at all. However, I'd like to find a medium similar to Advanced DMEM/F12 with a lower (only around 0.2-0.3 mM calcium concentration. The calcium concentration in Advanced DMEM/F12 is 1,05 mM).

The DMEM no calcium for example contains less ingredients (for instance proteins) than Advanced DMEM/F12 and is a "conventional" medium which requires 5-10% FBS supplement.

I'm looking for a medium with similar ingredients as Advanced reduced serum DMEM/F12 but no more than 0.3mM calcium , and which works without FCS supplementation or requires only very low FBS supplementation.

Hello,

I am Mahmuda, now I am working with DG44 cell culture. So far, my cell culture viability improved to 90%. However, before transfection, my cell viability decreased to 70%. I am very disappointed with the results of this culture.

Is there any suggestion or input that can be given so that I can solve this problem?

Then, is there any particular trick to do for this DG44 culture?

Thank you for the help

Hi everyone. I want to DNA isolate from transfected cell culture medium (transfected with lentivirus). I tried lots of kit but DNA cons and quality not very well and I didnt show at agarose gel.

For cell culture , if want to study about the protein or gene expression ? The treatment need to dilute with medium without fbs & pen strep or complete medium. What happen , if the treatment dilute with complete medium ?

Some types of cells was cultured by some medium contained with 2,4-D. To identify it's cell status, I make DAPI staining and the results make me confused.

Just as the pictures shows, the first one is DAPI staining, is the staining area represents the nuclei ?  It occupy a large proportion of the cell 

If anyone is familiar with the cell status and has any idea or suggestion, i would be so grateful, Much thanks !

Recently, we have been encountering challenges in our laboratory in achieving satisfactory mineralization of MC3T3-E1 cell line. We would like to determine the optimal concentration of Ascorbic Acid and β-Glycerophosphate to achieve the desired outcomes. Additionally, we are unsure whether we should initially culture for 8 days with Ascorbic Acid only and then for another 8 days with β-Glycerophosphate only. We appreciate your assistance in resolving these issues and thank you in advance for your collaboration.

Kind regards,

Antonio.

For transfection we have used 500uL of transfection complex for 1 well of 6 well plate. so total media added to each well was 2mL. According to this 100nM of sirNA and 100ug/mL of nanoparticle was added in the transfection complex (500uL). What is the ratio of SiRNA to media. (we have a stock of 100micro molar of siRNA )

Also what if we lower down the volume of media to 50uL. Then how to maintain the ratio of siRNA to media?

Hello everyone, I am having trouble with my cell culture. I have used the same cells in the passt, but now every time I thaw a batch (no matter if my own stocks or the original stocks of the lab) the cells are viable for a day or to, but as soon as I passage them the die. However, they don't die directly - they do adhere and start to proliferate, but then the bekom apoptotic. I've checked the medium and every thing else I can think of, and no one else is having trouble with the incubator. Does anyone have any trouble shouting ideas?

Thanks in advance!

Hello,

I've been trying to isolate exosomes from cell culture supernatant. I have spoken to other researchers who do exosome work and found that they don't use RIPA buffer to lyse the eVs when doing protein quantification (BCA) or western blots. They don't lyse the eVs at all. They claim that it degrades the eVs. However, in the literature, it seems common practice to use RIPA buffer or some type of lysis buffer. I have tried to find papers that support this claim but I haven't really found any. Just some papers comparing different lysis buffers.

Is the claim that RIPA or any lysis buffers isn't good for exosomes and characterizing them true? What to do instead?

Thank you for your time,

Britney

Hello fellow researchers,

i need some advise. I am trying to incubate some cells with a volatile substance and i am looking for ways to keep the substance from evaporating and keeping it in the 24well plate. I am using aluminum adhesive foil right now, but it seems to evaporate through the foil. Does anybody have an idea how i could seel my plate? Is there a foil with individual seeling for each well? I would be thankful for your experience!

I used to prepare DMEM-powdered media for the cell culture use. The last step of media preparation is media filtration using 0.2um filters. Meanwhile, in case of 0.2um filters shortage, is it possible to alternatively prepare a sterile media for the cell culture without using 0.2um filters. Like to use 0.45um filters and then autoclave the media to ensure the sterile condition of the prepared media?

I am working on a project in which we obtain lymphocytes through Ficoll density gradient separation and then culture them for ~3 weeks. Naturally, we add IL2 to our cell culture media. There are two practices that I have seen students do: one is to make a big batch of media with IL2 in it at the start of the experiment and use it over the 3 weeks (The media bottle is stored in the fridge at 4 Celsius. The second is to make the big batch of media without IL2 and only add IL2 to the culture flask when the media needs to be changed. Which approach do you think is better? (Note: in the second approach, the aliquots of IL2 undergo repeated freeze-thaw cycles. Since very little concentration of IL2 is needed in the culture (ng/mL), we do not make single-use aliquots as there would be a lot of them. We make aliquots with 100ug/mL concentration, thaw them upon need, use whatever is needed, and then freeze the rest for later use.)

Does anyone have good lab practices or experience of growing primary cells without antibiotics and anti-fungal?

Recently I ordered primary airway epithelial cells (adherent) from ATCC. They provided Anti-anti (penicillin + amphotericin B solution) along with basal media. so I prepared media with anti-anti solution and re suspend cells. so I observed cells were not attaching for 2 days and 3rd day there was contamination without change of media color. it was rod shaped bacteria with cells when i saw under microscope.

So when I complaint to ATCC, they suggested me some points. One of them was not to use anti-anti solution (antibiotics), as it can cause stress to primary cells.

Now I am planning to grow primary cells without adding antibiotics, But I am scared that there will be contamination and I dont want to lose cells (I have only single vial).

Any suggestions?

Hello everyone,

lately we experienced some problems with the performance of our NK92 cells, because they are performing way worse in cytotoxic assays than a few months ago. We thought this might either result from the NK92 cells being exhausted or resting. So we tested for the NK cell exhaustion markers DNAM-1 and NKG2a. Our NK92 cells were negative for DNAM-1 and positive for NKG2a, which might indicate that they`re in an inactive state.

Has anyone else experienced this before? Or does anyone know how to reverse exhaustion in NK92 cells?

*Medium + 100U/ml is given two-three times a week

Dear all,

I'm culturing several adherent cell lines. Recently, I noticed that I had dot-like particles in my cell cultures that have movement. Cell culture medium does not appear turbid. I suspected FBS contamination, so I started with new cells and a new batch of FBS, but the problem wasn't solved. I have attached the photos.

If anyone knows the problem, please reply.

Thank you so much, all.

Our lab buys 500 mL bottles of RPMI-1640 Medium and stores them at 4C. Oftentimes, for my experiments, I only need about 100 mL total. So, I will make RPMI-1640 + 10% FBS with 90 mL of media and 10 mL of FBS. Then, I always tightly wrap parafilm around the remaining 400 mL of media and put it back in 4C.

I work with cell culture of keratinocytes and from the second passage onwards I have to reduce the amount of calcium in the culture medium. Does anyone have a protocol on how to use the Chelex 100 resin (Bio-rad) to chelate calcium from a cell culture medium? Could you please share the protocol. The culture medium used is William medium E.

Thank you

Will someone have a protocol or reference on using Ficoll-400 to separate viable vs dead cells in suspension cultures? I am culturing Jukrat cells but will also be culturing DU4475s soon. Thanks

Hello everyone,

I renovate the cell list for the cells we store in -196 liquid nitrogen tank in our lab and I wonder what you include in your list for this. Cell name, passage number and the date I passage and store them in the tank are my elements for the list. What would you add beside these?

Thank you.

Our CO2 supply is outside in a cage and there is fairly long distance pipework going around the outside of the wall to the entry point in the wall of the lab. Since turning late Autumn/early winter, some of our cells are starting to look a bit odd. It seems to get worse the colder it gets. The incubators are reading 5% CO2 (so unlikely a leak), the temperature is correct too. They are newish incubators, only serviced recently (we are getting our own CO2 meter to check soon too). All reagents were replaced (several times). Two different people have had the same problem, so not user error either (both experienced users). Different batches of cells have been tried too. Its the first time Ive ever used a supply from outside, (its usually next to the incubator) so I was wondering if it had an effect on anything as Im running out of ideas. Many thanks

I have tried to achieve a separation of the nucleus and cytoplasm by using RLN buffer+ NP40 (0.2%), and then proceeding with GeneMATRIX universal RNA purification kit (the same used for total RNA extraction) and followed cell culture RNA purification protocol. I have managed a separation with this protocol in HeLa cells, but SH-SY5Y cells seem to be very sensitive. I have added the protocols and would appreciate any suggestions.

I saw these structures forming in my cell culture. Does anyone know what these are? Have you experienced such a situation before?

1. Take the vial with the cells and thaw it at 30 degrees in a water bath.

2. As soon as the culture is thawed and liquid, remove the vial from the water bath, and clean it using 70% ethanol. Take the 1 ml cell culture from the vial and add it to a 10 ml falcon containing 4 ml of complete Schneiders media (Complete S2 Media: 10% FBS, 1% Pen-Strep, 89% Schnieders Medium). Mix them nicely with pipetting.

3. Place the falcon, now with 5 ml components (1 ml of culture from stock and 4 ml fresh media) in a centrifuge at 100g for 10 min.

4. Remove the supernatant, which would contain DMSO, and then add fresh 5 ml of media (2.5 Fresh S2 Complete Media + 2.5 Conditioned S2 Media) and add this culture to the T-25 flask and let it incubate for 3-5 days before starting passaging.

Am I doing something wrong while I reculture the frozen stock?? Or is it alright and I should just clear out the dead cells using low centrifugation speed for 10 minutes or so?

I am growing small cell lung cancer suspension cells. How do I separate dead cells from live cells while splitting the cells after confluency in flask?

Does anyone have a schematic for hypoxic containers for irradiation experiments? We want to irradiate cells under hypoxia, and need containers for cell culture flasks that can be closed airtight in an hypoxic incubator, and taken to the irradiation equipment.

How to sterilize a polymer grafted with a photoinitiator before cell culture?

i am trying to culture the cells and see how these MEFs differentiate but each time i change the media the micromass layer(with 100,000 cells) is disrupted and i get to see them in a monolayer instead of being in a compact form.

I am looking for a mammalian cell media with a few specific supplements, but it is not available for purchase from any vendor. The lab-grade supplements are pretty expensive and are sold in either huge tubs or in tiny amounts. I thought to just buy the required supplements (L-arginine and L-lysine) in the pharmacy and add appropriate amount to my media. Do you think it is ok? I shouldn't list the source of supplements when it is time to publish? The pack size and purity of pharmacy-sold supplements seem to be suitable for my experiment.

Cheers,

Maria

Hello! The freezing protocol for MSCs I have been performing says to freeze 1 ml of cell suspension. I was told that in order to keep confluence, I must add only 1 ml to the pellet of cells and freeze that 1 ml in a criovial. However, my doubt is if instead of freezing in a criovial the 1 ml, I could divide by 3 criovial (around 330µl), increasing the stock of frozen cells.

Dear researchers kindly let me know is it will be feasible to culture blood cells in animal cell culture lab and put the isolated cells to grow in cell culture medium in the same CO2 incubator in which cancer cells are growing? Or it will create problems or have to put separately?

Needs your valuable suggestions

I've designed media for CHO cells, without Glutamine! But when they're in large scale, the cells were damaged and decreased their viability. What's your refer ?

Can anyone advise me on leader sequences to obtain recombinant proteins that are secreted by the cell culture? I should use mammalian cells for expression (from the Expi293 kit by ThermoFisher), and I would like my protein to be secreted. There is literature suggesting the use of the Ig-k chain or the CD33 sequence, but I can't find the reference coding sequence. Also, since the leader sequence would need to be added to a primer for PCR, is there a risk of it becoming too long?

Thank you for your help

I borrowed a bottle of HEK293T cell culture from someone else, but after one passage, the cell morphology became very strange and not round at all. May I ask why? We used T25 culture flasks and digested them with 500 microliters of pancreatic enzyme at 37 degrees Celsius for 30 seconds.

Hello Everyone, may I ask one question.

Caco2 cells were culture on hydrogel with different concentration. Color changes of medium was detected on some well after 3 weeks of cell culture. Can someone please give me a clue of what could be the main possibility?

Hi

I have be studying on bone derived MSC and in the begining I did passaging, ICC, freezing cells and defrosting cells. There was no problem but now my MSC cell cultures were infected two times and I could not find the reason.

I have suspected;

1. Last time I forgot to wash my cells before trypsinization but this affects only number of cell that I gained.

2. I have realized I was doing differentiation assay at the same time and cells in diffrentiation media were not affected from infection. In my diffrentiation media I was using gentamycine and in control media I used penicillin/streptomycin (Pen/Strep). Is this the reason or Pen/Strep does not work?

3.I recieved the cells on 6/12/2023 and I passaged them 16/12/2023 and I fed them on 19/12/2023. There was no problem and I would feed and passage them on 22/12/2023 but I have learnt they were infected?

I was checking them every feeding time is that enough or should I check them every day under microscope ? I do not want them they are infected or disturbed.

I will be so happy if you share your thoughts because I do not want to lose my cells because of infection third time?

Thank you

Hello everyone,

I would like to use platelets in a co-culture with other cells.

I was wondering which are the conditions to keep alive the platelets:

-since I am going to buy my platelets, I was wondering if they can be shipped on ice

-which buffer do I need to keep them alive during the shipment?

-I saw some papers where they were kept in culture with RPMI, do you have any suggestion/ideas about that?

Looking for your help

Thank you in advance

Hi, everyone.

I resently made the expression and purification of a GST recombinant protein. I am wondering whether this protein can have its function by added in the 293T or HeLa cell culture?

If not, any methods to make this protein have function?

Thanks!

Hi,

I have been trying to find the most appropriate protocol for myself to treat mouse splenocytes with fatty acids, but I am not sure what the most appropriate condition is for the experiment.

I have checked fatty acid levels in mouse plasma using mass spec and am trying to mimic the concentrations detected by mass spec. The plasma free fatty acids (FFA) was extracted using 90% methanol. The question is what is the best method to treat cells with different FAs if I want to mimic the plasma Fatty acid levels in culture?

I am considering between the two conditions below  for my experiment

1. Complex FA to BSA and use FA-albumin complex to treat the splencoytes for the assay

2. Dissolve FA in an appropriate solvent (i.e. DMSO, ethanol, etc) to make FFA solution.

If any of you could give me great advice/explanation, that would be really appreciated. Thanks for your help!

Kind Regards,

Takumi

How long cancer cell can survive in trypsin and complete media (1:6) solution in room temperature (bsc)?

Hello everyone,

I'm curently working on HaCaT cells which require low calcium medium to return to undifferentiate state. To do so, I need to chelate my FBS for 1h with chelex resin among other things.

Typical protocol suggests to chelate FBS, add to medium, then sterile filter the complete medium.

As only the FBS + chelex is not sterile and because filters are not that cheap, I rather filter sterilize a complete bottle of chelated FBS and then use it to complete my medium bottle.

Problem is the resin is clogging my 0.22µm filter, even 0.45µm get clogged before getting the 500mL of FBS passed through...

I was wondering if anyone had come across this problem and may have a handy solution.

- should I centrifuge the bottle before filtration and leave a dead volume ?

(i tried 5min 1500g but it didn't help much) maybe go higher ?

- maybe pass the FBS through a 80µm cellular sieve ?

- any ideas ?

Thanks for your time,

Philippe.

Hi!

I want to deproteinize a sample and then apply the resulting deproteinized product to cells in culture (PBMCs). Which method can I use for this? I thought about TCA, but I am not sure whether, even after neutralization, the sample would be toxic for the cells.

Thank you in advance.

Hello, is anyone doing cell culture for shrimp hemocytes? I have a problem with cell sorting; I cannot get the cell sorted out. I hypothesize that I used the L15 medium and did not adjust the osmolarity. Does anyone know about the L15 medium? Should we adjust the osmolarity of the L15 medium based on the osmolarity of shrimp hemocytes?

Dear all,

I recently encountered a problem with how to add exogenous ceramide to cell culture to monitor their biological effects. Currently, we want to add two ceramides to the cell culture： Ceramide (d18:0/24:0) and Glucosylceramide (plant), but we found it was only a little soluble in chloroform or DMF. These organic solvents cannot add 1% to cell culture which will also limit these ceramides' delivery to cells. Does have any better buffer that could form the lipid vesicle or stable suspension for subsequent cell treatment? Please the lipids experts or biologists give us some suggestions or advice, thank you very much!

I recently purchased a vial of Cynom-K1 cells. I grew them up over several weeks as they were of VERY poor viability when they showed up. Expanded them, froze back 35 vials, saved a couple million and did a DNA extraction. Amplified an intron from a region for which I had Cyno and Rhesus sequence to confirm that these were actually Cyno cells. To my surprise they were HUMAN. A warning to anyone getting in cells, make sure you know what you have before starting any work with them.