Cell Culture

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9 Hippocampal neuron attachment?
By Bian Liu · Johns Hopkins University

Karen Smith · University of Louisiana at Lafayette

sounds like good stuff for the neurons, so it is most likely an attachment issue. Those pre-coated coverslips available through VWR look cool. I might... [more]

sounds like good stuff for the neurons, so it is most likely an attachment issue. Those pre-coated coverslips available through VWR look cool. I might order some myself.

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43 What could be the reason for low recombinant protein yields from BL-21 E. coli cells?
By Arianna Celis · California State University, Los Angeles

Sharad Purohit · Georgia Regents University

what is the MW of the protein, ecoli system is not very reliable when the MW exceeds 60kD. also did you tested that the protein is not a inclusion bod... [more]

what is the MW of the protein, ecoli system is not very reliable when the MW exceeds 60kD. also did you tested that the protein is not a inclusion body.

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4 What amount of cells at a density of 2.0x10power5 is suitable for RNA harvest
By Stephen Karori · Egerton University

Stephen Karori · Egerton University

Sagar......thanks. Please elaborate how the procedure was carried out. If i harvest and put in liquid nitrogen immediately, will i achieve the same e... [more]

Sagar......thanks. Please elaborate how the procedure was carried out. If i harvest and put in liquid nitrogen immediately, will i achieve the same effect? will appreciate your answer as soon as you can

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1 Is there any differences in the cytokine responses between supernatants from unstimulated PBMCs and plasma?
By Fatoumatta Darboe · Medical Research Council (UK)

Peter Siska · Duke University

What do you mean by cytokine responses? And what kind of stimulation?

What do you mean by cytokine responses? And what kind of stimulation?

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60 RAW 264.7 culture issues
By Swetha Srinivasan · Georgia Institute of Technology

Eemin Tan · Queen Mary, University of London

Hi Juliane, thanks for getting back to me! Yes, I use the same carrier free RANKL. May I know what density you plate the RAW cells for differentiation... [more]

Hi Juliane, thanks for getting back to me! Yes, I use the same carrier free RANKL. May I know what density you plate the RAW cells for differentiation? I've tried 5k - 30k cells/ 0.32cm2 and TRAP stains were negative day 3 onwards =\ I'll thaw out some new cells to try next week. So far, like you've said I've used P5-11 but they've only proliferated. Could I clarify a few points- A. differentiating media is 20ng/ml RANKL in aMEM? B. Do you plate the cells in the differentiating media or leave to adhere then replace with differentiating media? Thanks!

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151 What are the best kits for RNA isolation?
By Danielle Smith · University of Cape Town

Alaaeldin Saleh · Qatar University

Qiagen is the best

Qiagen is the best

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2 Is 5-chloromethyl fluorescein diacetate suitable for GSH determination by flow cytometry?
By Etel Vieira · Universidade Federal dos Vales do Jequitinhonha e Mucuri

Etel Vieira · Universidade Federal dos Vales do Jequitinhonha e Mucuri

Hi Fernando, thank you! I'll try this. We have a FacsCanto with the violet laser.

Hi Fernando, thank you! I'll try this. We have a FacsCanto with the violet laser.

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22 Black dots like contamination in my cell cultures; in tightly adhered manner. What to do??
By Deepak Chhangani · Indian Institute of Technology Rajasthan

Sagar Gaikwad · Indian Institute of Advanced Research

I will suggest to start with fresh media, vial of Cell line, and other culture reagents.

I will suggest to start with fresh media, vial of Cell line, and other culture reagents.

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15 Shifting of cells from liquid nitrogen to -70C
By Priyanka Srivastava · Working under Dr. Gursaran Prasad Talwar

Raju Yadav · Sandor Proteomics

@neha singhal exactly neha , I observed the same , i think it because of the way we collected blood and separation , as in our study patients will be ... [more]

@neha singhal exactly neha , I observed the same , i think it because of the way we collected blood and separation , as in our study patients will be Immune suppressants , so some times drug toxicity matters

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7 Can I store the drug solution made in cell culture media?
By Bashir Yousef · China Pharmaceutical University

Neha Singhal · International Centre for Genetic Engineering and Biotechnology

Hello Bashir, i had almost a similar kind of question here, few days back , the only difference is my drugs are soluble in DMSO, but we make working ... [more]

Hello Bashir, i had almost a similar kind of question here, few days back , the only difference is my drugs are soluble in DMSO, but we make working concentrations, in cell media ( take a note that we use serum free media for this as Debashish Paul says). For reference you can visit this link https://www.researchgate.net/post/Should_serum_be_added_to_drug_dilutions_made_in_cell_culture_media2

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2 RAW cell ROS without LPS?
By Qaisra Choudhary · Kyung Hee University Medical Center

Neale Foxwell · University College London

The problem most people have with RAW 246.7 cells is that they grow them the wrong way....... and I am assuming that this is your problem. In my hand ... [more]

The problem most people have with RAW 246.7 cells is that they grow them the wrong way....... and I am assuming that this is your problem. In my hand I can passage the cells nearly 100 times and still get reproducible Nitric Oxide induction. Most people grow them on tissue culture plastic.....this is completely the wrong approach. Two reasons:- The cells adhere to the plastic very strongly and is is impossible to detach 100% of them. This causes problems as over trypsinisation will activate your cells to produce ROS thus you will see a higher background of ROS production in your control cells Some people choose to scrape the cells off the plastic....again this will affect the viability of the cells and AGAIN activate them to produce ROS...again you will see a higher background of ROS production in your control cells. What we do is simple....grow these cells in Techne Biological stirrer bottles designed specifically for suspension culture.....NO TRYPSINISATION REQUIRED, SAVE MONEY ON TC PLASTIC, SAVE MONEY ON PBS AND TRYPSIN..... and get healthy non activated cells for experiments. Hope this proves useful

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3 cis-ddp as a control in MTC assays, is it always necessary to perform such a control?
By Antonio Donaire · Universidad de Murcia

Aniko Huizer-Pajkos or Pajkos · Kolling Institute of Medical Research

Hi Antonio, I agree with Julie.

Hi Antonio, I agree with Julie.

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25 Which is more economical: coverslips or a specific haemocytometer cover?
By Triona O'Connell · Dublin City University

Stefan Schöffberger · Science Services GmbH

What do you all think about disposable coverslips? - If it is just to show how counting works it could be cheaper to use these with students. Dependin... [more]

What do you all think about disposable coverslips? - If it is just to show how counting works it could be cheaper to use these with students. Depending how often something breaks and how many counts the students should do... sure you want to show the good old haemocytometer as well.

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14 Can anyone please show me how to increase transfection efficiency when lipofectin 2000 was used on the cell line 3T3 or neuronal cell line GT1-7
By Kan Yang · Donghua University

Milagros Zavaleta · FARVET

Superfect reagent is an option, I used it to transfect other cell line (DF-1 cells) with plasmids of 6 Kb and it works well.

Superfect reagent is an option, I used it to transfect other cell line (DF-1 cells) with plasmids of 6 Kb and it works well.

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111 Cells detaching in culture?
By Neeraj K Satija · Defence Research and Development Organisation

Anbarasu Kumaraswamy · Indian Institute of Technology Madras

Water level in the incubator plays a critical role. Make sure enough water is there to maintain the humidity of the Incubator.

Water level in the incubator plays a critical role. Make sure enough water is there to maintain the humidity of the Incubator.

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28 MCF10A cells growing very rapidly, much more than normal, any ideas why?
By Michael Fenn · University of Florida

Lynn Thomas · Dalhousie University

I recently acquired MCF10A cells and also find them to be very fast growing. I use the same growth medium as you do. I think you should consider the... [more]

I recently acquired MCF10A cells and also find them to be very fast growing. I use the same growth medium as you do. I think you should consider the possibility that the fast growing phenotype is the appropriate one, since your cells were newly purchased from ATCC. The authentication procedures used at ATCC are known to be very reliable.

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24 What is the best way to separate human granulocytes from whole blood or buffy-coat? How long can neutrophils survive in culture media?
By Rocco Cantisani · Novartis Vaccines

Paige Lacy · University of Alberta

You can also lyse the RBCs after Ficoll sedimentation of granulocytes. Our lab routinely lyses RBCs after collecting the pellet from Ficoll centrifuga... [more]

You can also lyse the RBCs after Ficoll sedimentation of granulocytes. Our lab routinely lyses RBCs after collecting the pellet from Ficoll centrifugation, and uses ddH2O for this step (for no more than about 30 seconds). After this, we resuspend cells in 12 ml RPMI containing 5 mM EDTA to reduce clumping.

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New Does anybody have Agrobacterium rhizogenes with antibiotic resistance gene?
I need it to produce infinity root production.

I need it to produce infinity root production.

By Taha abd elrahman · Université de Sherbrooke

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29 We are having problems growing caco-2 cells, they grow very slowly . Can anyone give us tips on how to make them grow faster?
By Claudia Calvet · Fundação Oswaldo Cruz

Marius Hittinger · Universität des Saarlandes

Can you please define "very slowly" ? If you want a closed layer expressing high TEER-values on Transwells (for example) ... well ... this needs some ... [more]

Can you please define "very slowly" ? If you want a closed layer expressing high TEER-values on Transwells (for example) ... well ... this needs some time.

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15 A good protocol to purify monocytes for culture from human blood
By Gill Dealtry · Nelson Mandela Metropolitan University

Philippe Poncelet · BioCytex, Marseille, F (Stago group company)

Most previous suggestions look appropriate. If ever you use negative magnetic separation after density gradient (getting rid of all other PBMC subsets... [more]

Most previous suggestions look appropriate. If ever you use negative magnetic separation after density gradient (getting rid of all other PBMC subsets with lineage MAbs) take care to work at 4°C. In my past experience using Dynabeads, I realized that monocytes "like" beads in general (since they are opsonization-specialized cells) and they tend to engulf the beads quite non specifically. Then, you may loose quite a few during negative selection. Room Temperature is enough for that, so keep them cool ! I would not recommend using positive magnetic sorting (e.g. with CD14) since your monocytes may be full of ingested beads after the procedure and they also can have "bad digestion", as human beings eating stones. Always question which level of purity your experiment is needing. The recipie will differ enormously from 80% to 95% and even more significantly for 99%. Then you electronic cell-sorting (flow cytometry) can provide high purity but with a low recovery and high cost/efforts. Last, you may associate several technologies: density gradient, ii) negative enrichment (in the cold) and finally elect. cell-sorting. Good luck. PP P.S. James, could you give more on your (very interesting and new - for me) statement: "CD14 can be expressed on cells other than monocytes,"

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18 How to prepare a 0,2 M phosphate buffer (Na2HPO4-NaH2PO4), pH 6.4?
By Valeria Scagliotti · Università degli Studi di Milano-Bicocca

Trung Kien Nguyen · University of Incheon

You can make sodium phosphate buffer directly from purity sodium phosphate powder by go to sigma website and search for sodium phosphate buffer. Good ... [more]

You can make sodium phosphate buffer directly from purity sodium phosphate powder by go to sigma website and search for sodium phosphate buffer. Good luck

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5 HUVECS , passage 6
By Priya Gogia

Carla Casala · Hospital Sant Joan de Déu

Passage 6 is high for HUVECs. If you can use passage 2 or 3.

Passage 6 is high for HUVECs. If you can use passage 2 or 3.

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8 Fungal contamination in cell culture flask.
By Deepa Gandhi · National Environmental Engineering Research Institute

Neha Singhal · International Centre for Genetic Engineering and Biotechnology

These are just crystals. If they would have been fungal contamination , then by the end of 48 hrs you would have noticed increase in the mass. But i... [more]

These are just crystals. If they would have been fungal contamination , then by the end of 48 hrs you would have noticed increase in the mass. But i must say the picture is very clear.

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Question:
New Please suggest the cell line which expresses endogenous myocardin protein
Please suggest the cell line which expresses endogenous myocardin protein

Please suggest the cell line which expresses endogenous myocardin protein

By Arun Pradhan · Cincinnati Children's Hospital Medical Center

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30 Does PBS buffer have any effect on cell adhesion?
By Ruben Lopez-Cortes · Universidade de Vigo

Hongjuan Cui · Southwest University in Chongqing

PBS should not have any effect on it. try different buffer, and let's see what happened.

PBS should not have any effect on it. try different buffer, and let's see what happened.

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16 Where can I find triple negative breast cancer cell lines?
By Miguel Gutiérrez · Instituto Tecnológico y de Estudios Superiores de Monterrey

Karen Murray · University of Dundee

ATCC have a good concise array of Breast Cancer Triple Negative cell lines

ATCC have a good concise array of Breast Cancer Triple Negative cell lines

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82 Human cancer cell line culture contamination
By Céline Gonçalves · Universidade do Minho

Nirmala Jagadish · National Institute of Immunology

It is mycoplasma only. You can test with mycoplasma detection kit..

It is mycoplasma only. You can test with mycoplasma detection kit..

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1 What protocols do people use to prepare cell blocks from CSF samples?
By Chandra Krishnan · Dell Children's Medical Center of Central Texas

Jaleel Miyan · The University of Manchester

In the past we have used a method that might work for this. I dont see how you are going to get CSF+cells into parafin unless you are just isolating t... [more]

In the past we have used a method that might work for this. I dont see how you are going to get CSF+cells into parafin unless you are just isolating the cells by centrifugation and then embedding them. In our method we put fluid containing cells into methyl cellulose based culture medium and then you can freeze that as a block and cut it on a cryostat or freezing microtome. Works great for any cells/tissues in cultue but also for handling primary cells/tissues.

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About Cell Culture

Cell culture is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment.

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Cell Culture

About Cell Culture

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