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Harvesting cells


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#1 Push

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Posted 13 March 2014 - 09:34 AM

Hi

 

Can excess trypsinization kill cells during harvesting?

 

thank you 



#2 Tabaluga

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Posted 13 March 2014 - 09:46 AM

Yep, it can. You can check the cells under the microscope however to see when the majority are detached and when you need to stop the trypsin reaction by adding medium.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 Push

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Posted 14 March 2014 - 12:32 AM

I added Trypsin and I think it's too much. I had added 1 ml of trypsin for cells plated on a 6 well plate and didn't add medium to stop the reaction. But tried to wash the cells with PBS and I found a clump formation after the centrifugation. I checked the cells with viability dye and found all were dead after flowcytometry analysis. I prepared these cells after a transfection experiment.

 

Thank you very much 



#4 Tabaluga

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Posted 14 March 2014 - 08:09 AM

This could be your problem then, you could try other trypsinization conditions to find out. It's not just about how much trypsin is applied, but also about how long. How long did you live the trypsin on, and was it at 37 °C ?


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#5 rhombus

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Posted 17 March 2014 - 05:40 AM

Dear Push,

 

As previously stated over trypsinisation will cause damage/death of the cells. The variables are:

Concentration of the Trypsin

Length of exposure

Temperature

 

Most protocols indicate that the trypsin should be in contact with the cells for as short a time as possible. It should act as efficiently as possible which means that we warm up the trypsin solution to 37oC. We also wash the cell monolayer with PBS without Calcium and Magnesium. Both these divalent cations inhibit trypsin activity

 

You can also use less aggressive enzymes to sub culture cells i.e. Pronase, Elastase, Hyaluroniadase, collagenase etc

 

KIndest regards

 

Uncle Rhombus



#6 Push

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Posted 17 March 2014 - 06:48 AM

I had incubated the cells at 37 degrees for 5 minutes. 

Thank you very much  all of you, I started repeating the experiment today, by Thursday I will be able to give u all some feedback. and I will be taking these points stated here.

 

Thank you



#7 Push

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Posted 21 March 2014 - 05:01 AM

Hello everybody!

 

I repeated the transfection for my epithelial cell lines ( two cancer cell lines and a control normal epithelial cells line),  This time cells were all alive. The problem was with the trpsinization reaction as last time I didn't stop using serum. I id the transfection with pMAX-GFP and turbofectin 8. Unfortunately there was a very low level of transfection (27 % fluorescence)  in the normal control epithelial cell line and nothing in the cancer epithelial cell lines after FACS analysis. pMAX-GFP available at the lab is quite old, 2 years,Could not figure out the problem still. But in the protocol it is mentioned to use  Opti-Mem reduced medium, but I had to use serum free media at the time of my experiment.



#8 Push

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Posted 28 March 2014 - 07:40 AM

Hello All,

 

I have repeated the the transfection with cancer cells of epithelial origin (MCF -7, MDA-MB-231, BT-20) using Turbofectin 8 and with pRFP-C-RS and pmax-GFP but ended with a very low transfection efficiency. My cells were live but with no transfection.

 

Hope you all could help me



#9 bob1

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Posted 28 March 2014 - 10:42 PM

So - what did you do? - we need details for any sort of troubleshooting.



#10 Push

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Posted 24 April 2014 - 04:49 AM

 
Hi,
 
This is the protocol that I had followed.
 
Protocol for transient transfection (adherent cells) with Turbofectin 8 - Origene
 
1. Cell Plating 
a) On the day before transfection, plate cells at a density of 1-3 x 105 cells in 
complete growth medium per well of a 6-well plate to obtain 50-70% confluence 
on the following day. 
B) Incubate overnight. 
 
2. Complex formation (perform this step immediately before transfection) 
a) Dilute 1 μg of DNA in 250 uL of Opti-MEM I (Gibco 51985). Vortex gently. 
B) Add 3 uL of Turbofectin 8.0 to the diluted DNA (not the reverse order) and Pipet 
gently to mix completely. 
c) Incubate for 15 minutes at room temperature. 
 
3. Transfection in complete culture medium 
a) Add the mixture prepared in Step 2 dropwise to the cells. Gently rock the plate 
back-and-forth and from side-to-side to distribute the complex evenly. 
B) Incubate at 37°C for 24-48 hours. 
Note: With TurboFectin, no medium change is necessary. If you wish to remove the complex, 
remove the medium 4-24 hours post-transfection and replace with fresh complete medium. 
 
Thanks


#11 bob1

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Posted 24 April 2014 - 07:00 PM

That's a perfectly standard transfection procedure.  Have you checked that the plasmids are intact and did you isolate the plasmids using a commercial kit (which one)? A260:A280 ratios? 

 

Try adding the DNA and turbofectin to a smaller volume of medium, maybe about 50 ul/well.



#12 Push

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Posted 25 April 2014 - 03:48 AM

Yes, I checked the plasmids by agarose gel electrophoresis and they were intact. Also the ratio was ok. I am repeating the same experiment next week. I will try as you have suggested. More what's the best confluence that you would like to recommend for seeding of cells.

 

Thank you very much



#13 bob1

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Posted 25 April 2014 - 05:21 PM

Anywhere between 50 and 70% should be fine.






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