Trypsin and cell culture

Trypsin and cell culture

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I am doing an experiment where I have treat the cells with a drug and calculate their counts. I would like to know if is bad to trypsinize the cells in consecutive days i.e. twice within 48 hours. How much time do the cells need to attach again on the surface of the flask? I want to know if I can treat them with drug the same day I add trypsin for counting.


It would be stressful for cells to be trypsinized 24 hours after seeding. After cells are plated, there is a lag time to start growing. Perhaps, during the lag time, physiological state of cells is stabilized. They have to express proteins digested by trypsin and display on the surface etc. Then you could disturb the state again by trypsinizing cells again if you replate cells after 24 hours. I would not deny some cells are not affected by trypsin much, but you should be carful.

if I can treat them with drug the same day I add trypsin for counting.

It is not preferable, but in some cases, such a way is taken actually. If you could, you might want to give us more information about which cells and drugs you are going to use.


If you trypsinize cells to count and do not replate them, you do not need to worry about the cell condition, as WYSIWYG says.

It is not advisable to trypsinize the cells too often when you have to maintain them. However, in your case the situation is different.

Time required to attach depends on cell type. In any case while performing an experiment, your cells should not be already under stress. Therefore you should seed the flasks/culture plates with a smaller concentration of cells such that they reach around 60% confluence in 24 hours. 24 hours is generally considered to be enough for cells to recover from stress due to trypsinization. (You can have a look at this post for doubling times of some cell lines).

After doing the treatment you can count cells whenever you think it is fine; this actually depends on your experiment and you can treat the cells for the time that suits the drug's pharmacokinetcs. Trypsinizing is fine now because you won't be using these cells again.

However, there are methods to calculate cell numbers without trypsinizing. Assuming that your flask is homogeneous, you can choose a section of it and calculate cell numbers manually using microscope. You may also use software like ImageJ to make the task easier. GFP expressing cells will also make the task easier.

Might be considered "not answering the question" but:

Option 1: Depending on the type of cells you are using, you can use milder methods to detach cells. Example include Versene which is essentially PBS + EDTA. Milder still is to just use PBS that has no Mg or Ca (no chelation).

Option 2: Duplicate your setup - instead of two dishes - one with drug, one without, you make 4 dishes (dispensed from a common source - i.e. if your dish is a 6cm dish with 3ml media, you make 4.5x3mls of cells at the final dilution you want) each plate gets 3mls from the same source so you know they have the same number of cells. Two dishes you add drugs, two you don't. On the day you want to count, you take the replicate plates and count them. This way, you get the count without touching the final dishes at all.

As a side experiment, if you put those trypsinized cells back on two dish (and replace the drug media in the drug treated replicate), and you count all plates on the last day of your experiment, provided cells are not at 100% confluency, any difference you see from your trypsinized vs untrypsinized can be viewed as potential error that you would have introduced if you went through your original methodology.

Effect of Trypsin on Cell Volume and Mass

NORMAL cells stop growing in culture when they reach a certain saturation density 1–4 (contact inhibition of growth). The cells are arrested at the G1 phase of the cell cycle, during which DNA synthesis is repressed and division does not occur 5,6 . Whereas tumour cells are either less sensitive or insensitive to contact inhibition of growth 4 , normal cells can be rescued from this inhibition. Treatment with serum 1,7 , cell derived factors 8 , low concentrations of proteolytic enzymes 8,9 and a number of other agents 10 leads, after a certain time lag, to a resumption of DNA synthesis and mitosis. In conditions favourable for DNA synthesis and division, the size of a cell-whether normal or transformed-increases considerably 11 , but only tumour cells form colonies 12 . We now report that both normal and transformed cells immediately increase their volume and dry weight (mass) when exposed to a low dose of trypsin. This occurs when DNA synthesis is inhibited, and does not require new transcription trypsin is therefore directly affecting the cell surface.

Cell culture splitting questions--why do you centrifuge the cells after neutralizing trypsin? And having issues with new lab techniques. is it just me?

Hi r/biology, I'm new here. I just started a research assistant position at a basic science lab on cells and of course, it's tough feeling useless in the beginning b/c all the research residents & techs are busy with either learning new techniques (blots, PCRs, etc) or busy w/their own projects.

My biggest question though is in cell splitting--why do you centrifuge after trypsinizing cells? I have always learned in my previous lab position, that after trypsinization of cells, neutralizing tryspin w/media, you have to centrifuge it for like 4 mins or so. Then you discard the supernatant, remix the pellet into a new 10mL of media and then distribute into plates. The lab here doesn't centrifuge they just pipette up and down for 5 mins and then they just shoot the mix into the plates.

As for me, previously, I worked 2 years in a basic science stem cell lab 3 years ago, and the only thing I did really well was cell splitting/plating/maintaining cell cultures. My old supervisor was super anal about being sterile and clean and wiping everything down w/alcohol, always wearing gloves, always dispose things properly, whether you were pipetting media or something really biohazardous. Especially in cell culture techniques.

At my new lab, besides feeling useless, I noticed there are major differences in how they carry out experiments. For example, when they pipette in the hood, they discard the tips randomly into some corner of the hood instead of into some 50 mL tube (how I was taught) or a tips jar. Or when they remove the old media from a plate, they don't use a vacuum + pasteur pipette (burned to be sterile), but pipette instead. They don't spray their micropipettes w/EtOH as often when they move things in and out of the hood. I'm not sure if I'm just undergoing adjustment issues or someting..

It's frustrating because I'm the lowest person in the lab, I have no skills beyond cell culturing but I dont want to be put in charge of other people's cells yet. And they (understandingly) don't believe me when I said I used to centrifuge cells. There's more things but I feel that each time I ask such things, it's a bit annoying to them. Like "oh well this is the shortcut" and I used to be drilled to do it in a certain manner by my previous lab manager 2.5 yrs ago who was extremely skilled.

Trypsin and cell culture - Biology

Subculturing Human Umbilical Vein Endothelial Cells (HUVEC)

1. Hank's Balanced Salt Solution without Ca++ and Mg++ (HBSS), from Biowhittaker, catalogue #10-543.

2. Trypsin-Versene Mixture (0.5% trypsin - 0.02% EDTA), from Gibco, Cat. #610-5300 or Biowhittaker, Cat. #17-161. Dilute 1:1 with HBSS before use.

3. Nutrient Medium, consisting of 20% Fetal Calf Serum from Gibco, Cat. #200-6140, 80% Medium 199 (M199) buffered with 25mM HEPES from Biowhittaker, Cat. #12-118, and supplemented with fresh 2mM L-Glutamine (final concentration) from Biowhittaker, Cat. #17-605E, and with 100 U/ml K-Penicillin G with 100 mcg/ml Streptomycin Sulfate from Biowhittaker, Cat. #17-719R or Gibco, Cat. #600-5140. On the day of use, add 50 mcg/ml Endothelial Cell Growth Supplement (ECGS) from Biomedical Technologies, Inc., Cat. #BT-203, (See Protocol #2), and 100 mcg/ml Heparin from Sigma Chem. Co., Cat. #H-3933 (See Protocol #3).

4. 0.1% Gelatin, from Difco, Cat. #0143-02, dissolved in pyrogen-free, tissue culture grade water and autoclaved.

1. Coat empty tissue culture flasks (Corning or equivalent) with gelatin, draining and removing the excess. Allow the flasks to dry (at room temperature in a laminar flow hood).

2. Aspirate the medium from a flask of primary HUVEC (isolated from one or more umbilical cords) at or near confluence (see refs. 1 & 2).

3. Wash the monolayer with 5 ml of HBSS (volumes are given for a 75 cm 2 flask use 1/2 as much for 25 cm 2 flasks).

5. Very quickly, rinse the monolayer with 2 ml of the diluted trypsin-versene, allowing the surface to remain covered for 10-30 seconds.

7. Add another 2 ml of trypsin-versene to the flask.

8. Cap the flask and agitate it briefly.

9. Examine the flask under a microscope to determine that the cells have detached this usually occurs within 1-3 minutes (N.B.: monitor carefully at this stage to avoid excessive exposure to trypsin).

10. Add nutrient medium directly to the flask. The serum component will quench the activity of the trypsin. The volume added is determined according to the following scale.
Split 1 flask into 3 flasks (to be confluent in

5-6 days)
(N.B. : The 1:6 ratio is the most efficient in terms of the quantity of cells produced per number of feedings required.)

11. Distribute the diluted cell suspension into the gelatin pre-coated flasks.

12. Bring the final volume in each flask up to 10-12 ml with nutrient medium.

13. Incubate the flasks at 37°C in 5% CO 2 + 95% air and re-feed every 2-3 days.

  1. This procedure allows efficient amplification of endothelial cell number from primary cultures of human umbilical vein and other human vascular sources at relatively low passage number. Continued passage will eventually result in senescent changes and loss of useful replicative potential.
  2. This procedure is currently the standard method utilized in the Cell Culture Core Laboratory of the Vascular Research Division, Department of Pathology, Brigham and Women's Hospital, Boston, MA.

1. Gimbrone MA Jr., Shefton EJ and Cruise SA: Isolation and primary culture of endothelial cells from human umbilical vessels. Tissue Culture Association Manual 1978 4(2):813-818.

2. Gimbrone MA Jr: Culture of Vascular Endothelium. Chapter 1 in: Spaet T. (ed) Progress in Hemostasis and Thrombosis, Vol. III, Grune & Stratton, Inc., 1976 pp. 1-28.

3. Maciag T, Hover GA, Stemerman MB and Weinstein: Serial propagation of human endothelial cells in vitro. J Cell Biol 1981 91:420-428.

4. Thornton SC, Mueller SW and Levine EM: Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science 1983 222:623-625.

K. C./M. Gimbrone
7/1985 (5/19/00)
Vascular Research Division
Department of Pathology
Brigham & Women's Hospital
Boston, MA

Preparing Endothelial Cell Growth Supplement Stock Solution (5 mg/ml)

1. Endothelial cell mitogen (aka ECGS), obtained from Biomedical Technologies, Inc., Stoughton, MA, Cat. #BT-203

2. Hank's Balanced Salt Solution (HBSS), Cat. #10-543 from Biowhittaker

3. 1 sterile disposable plastic syringe (e.g., 20 ml) with or without a large-bore needle (without is preferred for safety reasons)

4. Sterile Millex 0.22 micron syringe filter units (Millipore, Cat. #SLGS02505 or #SLGP033RB)

5. 2 sterile culture tubes (plastic, disposable)

1. Use 50.0 mg of ECGS per liter of medium you plan to utilize within one week (see steps 9 and 10). Continue the procedure in a sterile hood.

2. For each liter of medium to be made, place 5 ml of HBSS at 37°C into the sterile bottle of ECGS.

3. Cap and swirl the bottle gently, avoiding bubbling.

4. Transfer the liquid to one of the sterile tubes.

5. Rinse the bottle with an additional 5 ml volume of HBSS and add it to the liquid in the tube.

6. Draw up the solution into a syringe.

7. If used, replace the needle with a sterile Millex 0.22 micron filter, and filter the solution into the second sterile tube.

8. Change the filter after every 20 ml to prevent filter overload.

9. Store this stock solution at 4°C. (DO NOT FREEZE). Also note that the activity of ECGS drops after it has been in solution for 7 - 10 days.

10. To feed subcultured human umbilical vein endothelial cells, dilute this 5 mg/ml ECGS stock solution 1:100 in 20% Fetal Calf Serum in M199 with 100 micrograms/ml Heparin (see Protocol #3) in HBSS on the day of feeding. (The activity of ECGS in nutrient medium deteriorates after approximately 48 hours.) Do not store medium supplemented with ECGS and heparin for more than two days.

K. C./M. Gimbrone
7/1985 (5/19/00)

Preparing a Stock Solution of Heparin (10 mg/ml).

1. Powdered heparin sodium salt (Grade 1, from porcine intestinal mucosa), Sigma, Cat. #H-3393.

2. Hank's Balanced Salt Solution (HBSS) from Biowhittaker, Cat. #10-543.

3. 1 sterile plastic disposable syringe (e.g. 20cc) with or without a large-bore needle (without is preferred for safety reasons).

4. Sterile Millex 0.22 micron syringe filter units (Millipore, Cat. #SLGS02505 or #SLGP033RB).

5. 2 sterile plastic culture tubes with caps (e.g. 50 ml).

1. Weigh out 100 mg of heparin per liter of nutrient medium to be made for use within the next 7 days.

2. In a sterile hood, add the heparin to HBSS at 37°C (10 ml HBSS per liter of medium to be made) in sterile tube cap and swirl.

3. If the heparin is slow to go into solution, vortex it briefly, then leave it in a 37°C water bath for 10 minutes.

4. Draw up the heparin sterilely with a syringe and needle.

5. Replace the needle, if used, with a 0.22 micron filter and collect the filtrate in another sterile tube.

6. See Methods: Steps 9 and 10 in Protocol #2. Heparin may not be subject to the same decrease of activity over time in solution, but is routinely treated the same way as the ECGS.

7. Dilute this stock solution of heparin 1:100 in 20% Fetal Calf Serum in M199 with 50 micrograms per ml of ECGS in HBSS for use in feeding subcultured HUVEC.

In general, it is easiest to make up larger volumes of 20% FCS in M199 with 2mM L-Glutamine, plus 100 U/ml of K-Penicillin G and 100 mg/ml of Streptomycin sulfate in advance, as this can be stored for 10 days or more at 4°C, then, simply dilute the ECGS and Heparin stock solutions 1:100 into the volume of medium to be used on a given day.

Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture

Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.


Infection with the wild-type PEDV…

Infection with the wild-type PEDV isolate but not with cell culture-adapted PEDV benefits…

The S protein determines trypsin…

The S protein determines trypsin dependency of PEDV propagation. (A) Schematic representation of…

Characterization of S protein activation.…

Characterization of S protein activation. (A) An entry assay was performed as described…

Mapping the genetic determinant for…

Mapping the genetic determinant for trypsin-enhanced PEDV entry. (A) Putative organization of class…

Substitution of arginine at position…

Substitution of arginine at position 890 results in reduced syncytium formation capacity of…

Detailed product information


Handling information

Each type of cell or cell line responds to Trypsin-EDTA for Primary Cells in a unique manner. For optimum results, continuously observe the cells during the dissociation process to prevent damage. For cell-specific information, please refer to the product sheet supplied with the cells or cell line.

  1. Bring the DPBS, the Trypsin-EDTA for Primary Cells, and the Trypsin Neutralizing Solution to room temperature before use. Warm the complete growth medium to 37°C prior to use with the cells.
  2. For each flask, carefully aspirate the spent media without disturbing the monolayer. If the cell culture medium contains serum, each flask should be rinsed with DPBS twice prior to adding the Trypsin-EDTA for Primary Cells.
  3. Using 1 to 2 mL for every 25 cm 2 , add the appropriate volume of trypsin-EDTA solution to each flask (e.g., each T-25 flask would be dissociated with 1 to 2 mL trypsin-EDTA).
  4. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells, and then aspirate the excess fluid off of the monolayer do not aspirate to dryness.
  5. Observe the cells under the microscope. When the cells pull away from each other and round up (typically within about 3 to 6 minutes), remove the flask from the microscope and gently tap the culture flask from several sides to promote detachment of the cells from the flask. Do not over-trypsinize as this will damage the cells.
    1. Some strongly adherent cell types, such as keratinocytes, may take much longer and may require trypsinization at 37°C.
    2. Some cell types may require more vigorous tapping.
    1. Do not over centrifuge cells as this may cause cell damage.
    2. After centrifugation, the cells should form a clean loose pellet.

    Quality control specifications

    Legal disclaimers

    The product is provided 'AS IS' and the viability of ATCC ® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid. Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

    This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

    While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

    This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

    Why add phenol red in trypsin? - (Apr/27/2005 )

    anybody can tell me that should we use phenol red contained in our trypsin-EDTA 0.05% for the purpose to trypsinize my keratinocyte monolayer cell line??

    You don't have to use phenol red in the trypsin, it just helps ensure that you have the right pH, as too high or too low a pH will do more damage to the cells when they are in a fraglie state, such as when they are being trypsinised.

    o, i see..the phenol red as a pH indicator as what u mentioned. thus, it gives the red color of the trypsin-EDTA. i'm wondering to know why they added in the phenol red in trypsin?? is that just giving the red colour. and to adjust the pH of the trypsin-EDTA.

    i tend to order trypsin-EDTA from Gibco,, but it contains phenol-red. will it do much problem to my adherent monolayer cell line.

    according to the fact most of the mediums used in cell culture contain phenol red too, i don't think that will poison your cells.

    you're right. that's an information for you too as bob said.

    fred, u meant it doenst matter if we have phenol red contained in our trypsin EDTA. ??

    stilll yet, wondering why some manufacturers put in phenol red in medium and trypsin EDTA since they can just neglect the addition. right. why is that??

    i agree with the fact it doesn't mattter if there's phenolred in trypsin edta.
    But i disagree with the fact phenol red (or other ph indicator) is negligible in medium. Most of the time, it doesn't deserve special thing except indicate pH, but for me i use it for detecting cell clones in a 96 well plate too and to check part of the health of cells.
    BTW, i think ph indicator is quite essential information in cell culture.

    Pheol red is used in the trypsin-EDTA and medium as a pH indicator only (at least as far as i know), it is really useful for a quick visual check of the pH of the solutions, if they are purple the pH is too high, and yellow if the pH is too low and in both cases should be discarded.

    Not all commercially made medium contains phenol-red, you can even buy DMEM without it. Phenol red is an estrogen analogue, that can (and does) mimic the action of adding estrogen to a cell line. If you are doing work that involves estrogen treatment, then use media and trypsin without phenol red.

    If you are really worried about it, you can buy powdered trypsin and make up the solution yourself.

    Thanks Fred and Bob, i guess it should be much useful basically for visual observation on cells conditions, at least it raises up color.

    phenol red contained in either medium or trypsin should be less enough in order not to affect cells growth. so, here can be concluded that no big deal using phenol red in small quantity either in medium or trypsin as it performs as indicator,and not to harm cells growth as main.

    Is Trypsin/EDTA a detergent??

    Trypsin is an enzyme that nibbles away at the cell's protein anchorage to the plate. EDTA chelates Mg ions, causing the cell to round up. Both in concert cause a cell to detech from the plate.

    Why 10% serum for cell culture? - (Jun/19/2008 )

    why we using 10 % serum in the media. what happens is we increase or decrease the percentage of the trypsin.

    % of serum is medium is depending on the cell type that you culture, I sometimes use 5%, 20% for different cells.
    changing trypsin concentration should not do anything too much in my opinion, but if you change it, you need to change the incubation time for detaching cell so it will not digest your cell.

    Trypsin will somehow introduce more stress to the cells. That is why we need to deactivate it by adding serum media.

    10% is just found to be minimal amount required to be good for most cell lines (Serum is expensive--and cruel too!). But many cell lines require higher serum, and many do just fine with lower. You can even get away without serum with certain cancer cell lines using some synthetic components added to medium.

    Serum is just used as a hotch-potch of survival factors that we have not yet identified, but required for cell growth and survival.

    Trypsin damages cytoplasmic membrane, so you want to keep it to a minimum in concentration/incubation, immediately quenching it with serum once your purpose is served.

    10% Serum. what does this actually mean?

    Please put the following serums in order of quality?

    British, European, New Zealand, Australian, North American, South American?

    My point is that 10% serum from one country of origin is WORTH PROBABLY 70-100% from another. this is related also to the price:-

    £25, £45-£50, £250, £180, £120, £100. please fit the price to the above country of origin?

    10% Serum. what does this actually mean?

    Please put the following serums in order of quality?

    British, European, New Zealand, Australian, North American, South American?

    My point is that 10% serum from one country of origin is WORTH PROBABLY 70-100% from another. this is related also to the price:-

    £25, £45-£50, £250, £180, £120, £100. please fit the price to the above country of origin?

    I believe pricing has to do with the GARANTEED absence of a certain disease in a country for a certain amount of time. 10% usually means a part FCS added to 9 parts medium. As to the quality quenstion: in my opinion it is more important to have a steady supply of FCS with little variation in quality/components/source.

    10% Serum. what does this actually mean?

    Please put the following serums in order of quality?

    British, European, New Zealand, Australian, North American, South American?

    My point is that 10% serum from one country of origin is WORTH PROBABLY 70-100% from another. this is related also to the price:-

    £25, £45-£50, £250, £180, £120, £100. please fit the price to the above country of origin?

    I believe pricing has to do with the GARANTEED absence of a certain disease in a country for a certain amount of time. 10% usually means a part FCS added to 9 parts medium. As to the quality quenstion: in my opinion it is more important to have a steady supply of FCS with little variation in quality/components/source.

    Just for information: You are correct about NZ being the only BSE free country in the world

    The quality of NZ serum is the BEST IN THE WORLD. We do the following tests:

    Induction of iNOS Enzyme
    Oxygen consumption
    cGMP content of RASMC
    Cytochrome C concentration within J744 and Jurkats
    Oxidation/Reduction states of mitochondrial enzyme complexes
    Sodium Channel expression in transfected HEK cells


    i.e. Greater number of channels expressed, 80% greater induction of iNOS, greater oxygen consumption etc etc etc.

    There are even differences between NZ batches. thus we BATCH TEST to make sure that the results we get are consistant to that we got 5/10/15 years ago.

    Trypsin and Cell Dissociation Reagents

    Biological Industries’ trypsin and cell dissociation solutions are widely used for removing adherent cells from a culture surface. Choosing the right dissociation reagent and concentration depends on the cell type as well as the age of the cells in culture. BI provides a wide variety of trypsin solutions, which are prepared from porcine parvovirus-tested and mycoplasma-tested materials, in addition to chemically-defined alternatives.

    Looking for an animal-free alternative? Recombinant Trypsin Solutions - The best replacement for trypsin.

    BI’s Recombinant Trypsin Solutions are animal component-free, defined cell dissociation enzymes that replace porcine trypsin and avoid animal-related variability and contamination. Recombinant Trypsin Solutions are ideal for dissociating attachment-dependent cells in both serum-containing and serum-free conditions and can be directly substituted for trypsin without protocol changes. These solutions are also optimized for sensitive cell types, a critical characteristic in preventing harsh effects for downstream processing.

    A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow

    We explain a protocol for straightforward isolation and culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) to supply researchers with a method that can be applied in cell biology and tissue engineering with minimal requirements. Our protocol is mainly on the basis of the frequent medium change in primary culture and diminishing the trypsinization time. Mouse mesenchymal stem cells are generally isolated from an aspirate of BM harvested from the tibia and femoral marrow compartments, then cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) for 3 h in a 37 degrees C-5% CO(2) incubator. Nonadherent cells are removed carefully after 3 h and fresh medium is replaced. When primary cultures become almost confluent, the culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid for 2 min at room temperature (25 degrees C). A purified population of MSCs can be obtained 3 weeks after the initiation of culture.