Hello!
I am doing research with huvec and I have conducted huvec wound healing assay four times with 6 well plates.
I studied that gelatin coating should be done before working with huvec,
but it seemed that I did not coat 6 well plates with gelatin at the first assay.
I remebered coating gelatin at the rest of the assays. Despite such difference i could get highly consistent results.
So can I conclude that absence of gelatin coating does not matter in huvec wound healing assay?
I also want to learn whether amount of gelatin could affect wound clousre
rate.From first well to last 6 well, I reuse same 1ml gelatin by pouring 1ml
gelatin, shaking and suctioning again by pipette and pouring it to a next well
So I am concerned that more gelatin is coated on first three
sicontrol-treated wells
than next three 'specific gene silencing siRNA'-treated wells considering loss of gelatin during reuse.
Thank you! I expect great answers.
Any answers?
Dear Lee,
Generally, the plates are coated with Gelatin 0,2% and sometimes 1% for about 30 minutes to 40 minutes.
The following paper discusses coating by gelatin:
Biotech Histochem. 1992 Jul;67(4):241-50.
A comparison of substrates for human umbilical vein endothelial cell culture.
Smeets EF1, von Asmuth EJ, van der Linden CJ, Leeuwenberg JF, Buurman WA.
Author information
Abstract
We have studied culture conditions which facilitate the growth of stable, non-proliferating, human umbilical vein endothelial cell (HUVEC) monolayers. Gelatin and fibronectin coatings, with or without glutaraldehyde cross-linking, on both plastic and glass were investigated for initial attachment of HUVEC and growth characteristics. The presence during culture of intercellular (IC) junctions demonstrated by silver staining, expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and maintenance of a cobblestone appearance of HUVEC monolayers were assessed over time. Glutaraldehyde cross-linked fibronectin and gelatin coatings on glass and glutaraldehyde cross-linked gelatin or untreated fibronectin coatings on plastic served as good substrates for short term culture. Long term (20 days) cultures of HUVEC which maintained silver and PECAM-1 staining of IC junctions and a cobblestone appearance could be achieved if glutaraldehyde cross-linked gelatin coatings on glass were used as substrates.
Specific procedure for coating by gelatin:
The procedure .
1. Prepare 1mg/ml gelatin stock solution, dissolve by incubating in 37 degree water bath, filter it (to remove any floating materials) and finally autoclave it.
2.In the day of the experiment, coat the T-25 flask with aprox. 2ml of 1mg/ml gelatin (it should cover the surface of the flask) in side the hood and keep it for 1hr inside the hood.
3. After 1hr remove excess gelatin solution and use the flask for tissue culture.
The following is a protocol with collagen:
• Culture medium
M199 “stock”:
- M199 Earle 10X 100 ml
- Distilled water 900 ml
M199 “full”:
- Glutamine 0.2 M 1 ml
- HEPES 1 M 1.5 ml
- NaHCO3 7.5 % (w/v) 1.8 ml
- Penicilline/streptomycine (10000/10000) 1 ml
- FCS (heated at 56°C and centrifuged) 20 ml
- M199 “stock” up to 100 ml
Conservation at +4°C up to 1 week.
II- Cell culture
• The day before the manipulation:
- Coat the Petri dishes (35 mm; 7 dishes/cord) with 1 drop of fibronectin
- Bring at the maternity 3 recipients containing 50 ml of conservation buffer for cords
• The day of the manipulation:
- Fill up a recipient with ~ 500 ml of physiologic serum and maintain at 37 °C (“bain-marie”)
- Under the hood, prepare (for 1 cord):
¾ 2 syringes of 50 ml, 1 syringe of 30 ml and 1 syringe of 10 ml
¾ 1 surgical clamping clip, 2 cannulas, string, sterile compresses, sterile scalpels and
sterile gloves
- Spread on the working area under the hood: 1 aluminium foil, 1 paper sheet and the
“sterile envelope” of gloves (in this order)
3
• Cord manipulation and culture:
- Tidily cut the 2 ends of the cord with scalpel
- Introduce a cannula at each extremity of the vein (the widest vessel) and tightly maintain
it with string
- Wash the cord with PBS using the syringe of 50 ml
- Inject the collagenase (0.2 %) at one end of the vein using the syringe of 30 ml
- When leaking out the other end, tightly clamp it with the surgical clip
- Maintain the syringe and protect the cord’s ends with “clean” aluminium foil
- Incubate the cord 10 min in the physiologic serum at 37 °C
- Upon the sterile envelope of gloves, gently squeeze the cord
- Fill up a sterile falcon of 50 ml with 10 ml of “full” culture medium
- Collect the cells in this falcon by washing the vein with 40 ml of PBS
- Centrifuge at 900 rpm for 10 min
- Carefully discard the supernatant and suspend cells in ~14 ml of “full” culture medium
- Dissociate the cells (aspiration and repulsing X 3) with the syringe of 30 ml with needle
- Add 2 ml of the cell suspension in each fibronectin-coated Petri dish
Cultures are placed in a humidified 95% air and 5% CO2 atmosphere at 37° C.
The following day, non-adherent cells are removed by changing the culture medium. The culture medium is changed every two days and confluency is typically achieved in 6-8 days with “cobblestone appearance” in optical microscopy (~ 1.10 6
cells/dish).
Hoping this will be helpful,
Rafik
Dear Lee,
Generally, the plates are coated with Gelatin 0,2% and sometimes 1% for about 30 minutes to 40 minutes.
The following paper discusses coating by gelatin:
Biotech Histochem. 1992 Jul;67(4):241-50.
A comparison of substrates for human umbilical vein endothelial cell culture.
Smeets EF1, von Asmuth EJ, van der Linden CJ, Leeuwenberg JF, Buurman WA.
Author information
Abstract
We have studied culture conditions which facilitate the growth of stable, non-proliferating, human umbilical vein endothelial cell (HUVEC) monolayers. Gelatin and fibronectin coatings, with or without glutaraldehyde cross-linking, on both plastic and glass were investigated for initial attachment of HUVEC and growth characteristics. The presence during culture of intercellular (IC) junctions demonstrated by silver staining, expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and maintenance of a cobblestone appearance of HUVEC monolayers were assessed over time. Glutaraldehyde cross-linked fibronectin and gelatin coatings on glass and glutaraldehyde cross-linked gelatin or untreated fibronectin coatings on plastic served as good substrates for short term culture. Long term (20 days) cultures of HUVEC which maintained silver and PECAM-1 staining of IC junctions and a cobblestone appearance could be achieved if glutaraldehyde cross-linked gelatin coatings on glass were used as substrates.
Specific procedure for coating by gelatin:
The procedure .
1. Prepare 1mg/ml gelatin stock solution, dissolve by incubating in 37 degree water bath, filter it (to remove any floating materials) and finally autoclave it.
2.In the day of the experiment, coat the T-25 flask with aprox. 2ml of 1mg/ml gelatin (it should cover the surface of the flask) in side the hood and keep it for 1hr inside the hood.
3. After 1hr remove excess gelatin solution and use the flask for tissue culture.
The following is a protocol with collagen:
• Culture medium
M199 “stock”:
- M199 Earle 10X 100 ml
- Distilled water 900 ml
M199 “full”:
- Glutamine 0.2 M 1 ml
- HEPES 1 M 1.5 ml
- NaHCO3 7.5 % (w/v) 1.8 ml
- Penicilline/streptomycine (10000/10000) 1 ml
- FCS (heated at 56°C and centrifuged) 20 ml
- M199 “stock” up to 100 ml
Conservation at +4°C up to 1 week.
II- Cell culture
• The day before the manipulation:
- Coat the Petri dishes (35 mm; 7 dishes/cord) with 1 drop of fibronectin
- Bring at the maternity 3 recipients containing 50 ml of conservation buffer for cords
• The day of the manipulation:
- Fill up a recipient with ~ 500 ml of physiologic serum and maintain at 37 °C (“bain-marie”)
- Under the hood, prepare (for 1 cord):
¾ 2 syringes of 50 ml, 1 syringe of 30 ml and 1 syringe of 10 ml
¾ 1 surgical clamping clip, 2 cannulas, string, sterile compresses, sterile scalpels and
sterile gloves
- Spread on the working area under the hood: 1 aluminium foil, 1 paper sheet and the
“sterile envelope” of gloves (in this order)
3
• Cord manipulation and culture:
- Tidily cut the 2 ends of the cord with scalpel
- Introduce a cannula at each extremity of the vein (the widest vessel) and tightly maintain
it with string
- Wash the cord with PBS using the syringe of 50 ml
- Inject the collagenase (0.2 %) at one end of the vein using the syringe of 30 ml
- When leaking out the other end, tightly clamp it with the surgical clip
- Maintain the syringe and protect the cord’s ends with “clean” aluminium foil
- Incubate the cord 10 min in the physiologic serum at 37 °C
- Upon the sterile envelope of gloves, gently squeeze the cord
- Fill up a sterile falcon of 50 ml with 10 ml of “full” culture medium
- Collect the cells in this falcon by washing the vein with 40 ml of PBS
- Centrifuge at 900 rpm for 10 min
- Carefully discard the supernatant and suspend cells in ~14 ml of “full” culture medium
- Dissociate the cells (aspiration and repulsing X 3) with the syringe of 30 ml with needle
- Add 2 ml of the cell suspension in each fibronectin-coated Petri dish
Cultures are placed in a humidified 95% air and 5% CO2 atmosphere at 37° C.
The following day, non-adherent cells are removed by changing the culture medium. The culture medium is changed every two days and confluency is typically achieved in 6-8 days with “cobblestone appearance” in optical microscopy (~ 1.10 6
cells/dish).
Hoping this will be helpful,
Dear Lee,
Generally, the plates are coated with Gelatin 0,2% and sometimes 1% for about 30 minutes to 40 minutes.
The following paper discusses coating by gelatin:
Biotech Histochem. 1992 Jul;67(4):241-50.
A comparison of substrates for human umbilical vein endothelial cell culture.
Smeets EF1, von Asmuth EJ, van der Linden CJ, Leeuwenberg JF, Buurman WA.
Author information
Abstract
We have studied culture conditions which facilitate the growth of stable, non-proliferating, human umbilical vein endothelial cell (HUVEC) monolayers. Gelatin and fibronectin coatings, with or without glutaraldehyde cross-linking, on both plastic and glass were investigated for initial attachment of HUVEC and growth characteristics. The presence during culture of intercellular (IC) junctions demonstrated by silver staining, expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and maintenance of a cobblestone appearance of HUVEC monolayers were assessed over time. Glutaraldehyde cross-linked fibronectin and gelatin coatings on glass and glutaraldehyde cross-linked gelatin or untreated fibronectin coatings on plastic served as good substrates for short term culture. Long term (20 days) cultures of HUVEC which maintained silver and PECAM-1 staining of IC junctions and a cobblestone appearance could be achieved if glutaraldehyde cross-linked gelatin coatings on glass were used as substrates.
Specific procedure for coating by gelatin:
The procedure .
1. Prepare 1mg/ml gelatin stock solution, dissolve by incubating in 37 degree water bath, filter it (to remove any floating materials) and finally autoclave it.
2.In the day of the experiment, coat the T-25 flask with aprox. 2ml of 1mg/ml gelatin (it should cover the surface of the flask) in side the hood and keep it for 1hr inside the hood.
3. After 1hr remove excess gelatin solution and use the flask for tissue culture.
The following is a protocol with collagen:
• Culture medium
M199 “stock”:
- M199 Earle 10X 100 ml
- Distilled water 900 ml
M199 “full”:
- Glutamine 0.2 M 1 ml
- HEPES 1 M 1.5 ml
- NaHCO3 7.5 % (w/v) 1.8 ml
- Penicilline/streptomycine (10000/10000) 1 ml
- FCS (heated at 56°C and centrifuged) 20 ml
- M199 “stock” up to 100 ml
Conservation at +4°C up to 1 week.
II- Cell culture
• The day before the manipulation:
- Coat the Petri dishes (35 mm; 7 dishes/cord) with 1 drop of fibronectin
- Bring at the maternity 3 recipients containing 50 ml of conservation buffer for cords
• The day of the manipulation:
- Fill up a recipient with ~ 500 ml of physiologic serum and maintain at 37 °C (“bain-marie”)
- Under the hood, prepare (for 1 cord):
¾ 2 syringes of 50 ml, 1 syringe of 30 ml and 1 syringe of 10 ml
¾ 1 surgical clamping clip, 2 cannulas, string, sterile compresses, sterile scalpels and
sterile gloves
- Spread on the working area under the hood: 1 aluminium foil, 1 paper sheet and the
“sterile envelope” of gloves (in this order)
3
• Cord manipulation and culture:
- Tidily cut the 2 ends of the cord with scalpel
- Introduce a cannula at each extremity of the vein (the widest vessel) and tightly maintain
it with string
- Wash the cord with PBS using the syringe of 50 ml
- Inject the collagenase (0.2 %) at one end of the vein using the syringe of 30 ml
- When leaking out the other end, tightly clamp it with the surgical clip
- Maintain the syringe and protect the cord’s ends with “clean” aluminium foil
- Incubate the cord 10 min in the physiologic serum at 37 °C
- Upon the sterile envelope of gloves, gently squeeze the cord
- Fill up a sterile falcon of 50 ml with 10 ml of “full” culture medium
- Collect the cells in this falcon by washing the vein with 40 ml of PBS
- Centrifuge at 900 rpm for 10 min
- Carefully discard the supernatant and suspend cells in ~14 ml of “full” culture medium
- Dissociate the cells (aspiration and repulsing X 3) with the syringe of 30 ml with needle
- Add 2 ml of the cell suspension in each fibronectin-coated Petri dish
Cultures are placed in a humidified 95% air and 5% CO2 atmosphere at 37° C.
The following day, non-adherent cells are removed by changing the culture medium. The culture medium is changed every two days and confluency is typically achieved in 6-8 days with “cobblestone appearance” in optical microscopy (~ 1.10 6
cells/dish).
Hoping this will be helpful,
Dear Rafik Karaman,
Thank you so much for your great answers!
I will keep your answers for useful information!
But honestly, what I asked were whether lack of gelatin coating does not matter with huvec wound healing assay and about the reuse of gelatin. The detailed information are described on my question above.
I appreciated for your help again.
Dear Lee,
To judge from your experiment; no need to gelatin at all, and variable concentrations of gelatin are good enough to do the same job. However, I supplied you with some papers to show you that researchers are generally using at least 0.2% gelatin.
Best wishes,
Rafik
Dear Rafik,
Thank you so much!
Your answers are great help for me.
I always use 2% geltin. Thank you for confirmation.
I want to make sure about some of your answers
because My ability of understanding english is not
very good.
You mean that gelatin coating is not necessary but recommended for huvec wound healing assay and
different amounts of gelatin between 6wells due to gelatin loss
(3wells-siControl treated, Other 3 wells-siRNA treated) do not
matter right?
Sorry for bothering you for additional questions.
Hope for your happy new year!
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