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Macrophage migration Assay using the transwell system?
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+ Monocytes
+ Thp-1
+ Macrophage
+ Trypsin
+ Plating
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Mary Gallagher
Macrophage migration Assay using the transwell system?
Hi Yvonne,
Thank you you for the suggestions!! yes with the THP-1 I am checking for them in the medium in the bottom well. I am using a 8um pore membrane.
I thought the M0 (adherent) cells would fall through and adhere to the bottom of the well; I think that's where I might have gone wrong and missed them!
Is there an additional factor to PMA you suggest to differentiate them on the membrane?
Thank you you for the suggestions!! yes with the THP-1 I am checking for them in the medium in the bottom well. I am using a 8um pore membrane.
I thought the M0 (adherent) cells would fall through and adhere to the bottom of the well; I think that's where I might have gone wrong and missed them!
Is there an additional factor to PMA you suggest to differentiate them on the membrane?
Hi Sehar, Yes, they would be on the bottom side of your filter. You are using a good differentiation model but it's going to be tough. I never had good luck with THPs after differentiation. Have you considered using Mouse bone marrow derived macrophages or a different macrophage cell line? You can deattach them with a special protocol and plate them in equal amounts in the chambers, let them get adherent for 2-4h and start transmigration than. That works fairly well and you'd have better control over cell numbers. Just an idea. Good luck. Yvonne
Hi Sehar, Yes, they would be on the bottom side of your filter. You are using a good differentiation model but it's going to be tough. I never had good luck with THPs after differentiation. Have you considered using Mouse bone marrow derived macrophages or a different macrophage cell line? You can deattach them with a special protocol and plate them in equal amounts in the chambers, let them get adherent for 2-4h and start transmigration than. That works fairly well and you'd have better control over cell numbers. Just an idea. Good luck. Yvonne
THP-1s as you mentioned are non-adherent. I assume you checked the medium in the lower chamber for cells. Is that correct? As soon as you have adherent cells you need to check the lower side of your membrane. Adherent cells will migrate through the pores but not deattach, meaning they are just on the other (bottom side) of your filter. For this you need to swipe the top inside with a cotton tip, fix the filter, label nuclei with DAPI and image the bottom of your filter.
THPs might not like being differentiated on a filter, so, you might have an additional factor. And as soon as cells are adherent, you also might need to consider a different pore size. It is tough for a 40-50um cell to squeeze through 3/5um pores. 8um might be needed. I hope that helps. Yvonne
THP-1s as you mentioned are non-adherent. I assume you checked the medium in the lower chamber for cells. Is that correct? As soon as you have adherent cells you need to check the lower side of your membrane. Adherent cells will migrate through the pores but not deattach, meaning they are just on the other (bottom side) of your filter. For this you need to swipe the top inside with a cotton tip, fix the filter, label nuclei with DAPI and image the bottom of your filter.
THPs might not like being differentiated on a filter, so, you might have an additional factor. And as soon as cells are adherent, you also might need to consider a different pore size. It is tough for a 40-50um cell to squeeze through 3/5um pores. 8um might be needed. I hope that helps. Yvonne
Yvonne Baumer Hi Yvonne, your suggestions for macrophage invasion are quite relevant. Can you share a protocol that works? i am just starting out with macrophage biology and any tip would be helpful. Thank you.
Yvonne Baumer Hi Yvonne, your suggestions for macrophage invasion are quite relevant. Can you share a protocol that works? i am just starting out with macrophage biology and any tip would be helpful. Thank you.
THP-1s as you mentioned are non-adherent. I assume you checked the medium in the lower chamber for cells. Is that correct? As soon as you have adherent cells you need to check the lower side of your membrane. Adherent cells will migrate through the pores but not deattach, meaning they are just on the other (bottom side) of your filter. For this you need to swipe the top inside with a cotton tip, fix the filter, label nuclei with DAPI and image the bottom of your filter.
THPs might not like being differentiated on a filter, so, you might have an additional factor. And as soon as cells are adherent, you also might need to consider a different pore size. It is tough for a 40-50um cell to squeeze through 3/5um pores. 8um might be needed. I hope that helps. Yvonne
THP-1s as you mentioned are non-adherent. I assume you checked the medium in the lower chamber for cells. Is that correct? As soon as you have adherent cells you need to check the lower side of your membrane. Adherent cells will migrate through the pores but not deattach, meaning they are just on the other (bottom side) of your filter. For this you need to swipe the top inside with a cotton tip, fix the filter, label nuclei with DAPI and image the bottom of your filter.
THPs might not like being differentiated on a filter, so, you might have an additional factor. And as soon as cells are adherent, you also might need to consider a different pore size. It is tough for a 40-50um cell to squeeze through 3/5um pores. 8um might be needed. I hope that helps. Yvonne
Hi Yvonne,
Thank you you for the suggestions!! yes with the THP-1 I am checking for them in the medium in the bottom well. I am using a 8um pore membrane.
I thought the M0 (adherent) cells would fall through and adhere to the bottom of the well; I think that's where I might have gone wrong and missed them!
Is there an additional factor to PMA you suggest to differentiate them on the membrane?
Thank you
Sehar :)
Hi Yvonne,
Thank you you for the suggestions!! yes with the THP-1 I am checking for them in the medium in the bottom well. I am using a 8um pore membrane.
I thought the M0 (adherent) cells would fall through and adhere to the bottom of the well; I think that's where I might have gone wrong and missed them!
Is there an additional factor to PMA you suggest to differentiate them on the membrane?
Thank you
Sehar :)
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Hi Sehar,
Yes, they would be on the bottom side of your filter. You are using a good differentiation model but it's going to be tough. I never had good luck with THPs after differentiation. Have you considered using Mouse bone marrow derived macrophages or a different macrophage cell line? You can deattach them with a special protocol and plate them in equal amounts in the chambers, let them get adherent for 2-4h and start transmigration than. That works fairly well and you'd have better control over cell numbers. Just an idea.
Good luck.
Yvonne
Hi Sehar,
Yes, they would be on the bottom side of your filter. You are using a good differentiation model but it's going to be tough. I never had good luck with THPs after differentiation. Have you considered using Mouse bone marrow derived macrophages or a different macrophage cell line? You can deattach them with a special protocol and plate them in equal amounts in the chambers, let them get adherent for 2-4h and start transmigration than. That works fairly well and you'd have better control over cell numbers. Just an idea.
Good luck.
Yvonne
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Hi,
It could be an option.
thank you for your suggestions :)
Hi,
It could be an option.
thank you for your suggestions :)
More
VOTE
Hello Sehar,
THP-1s as you mentioned are non-adherent. I assume you checked the medium in the lower chamber for cells. Is that correct?
As soon as you have adherent cells you need to check the lower side of your membrane. Adherent cells will migrate through the pores but not deattach, meaning they are just on the other (bottom side) of your filter. For this you need to swipe the top inside with a cotton tip, fix the filter, label nuclei with DAPI and image the bottom of your filter.
THPs might not like being differentiated on a filter, so, you might have an additional factor. And as soon as cells are adherent, you also might need to consider a different pore size. It is tough for a 40-50um cell to squeeze through 3/5um pores. 8um might be needed.
I hope that helps.
Yvonne
Hello Sehar,
THP-1s as you mentioned are non-adherent. I assume you checked the medium in the lower chamber for cells. Is that correct?
As soon as you have adherent cells you need to check the lower side of your membrane. Adherent cells will migrate through the pores but not deattach, meaning they are just on the other (bottom side) of your filter. For this you need to swipe the top inside with a cotton tip, fix the filter, label nuclei with DAPI and image the bottom of your filter.
THPs might not like being differentiated on a filter, so, you might have an additional factor. And as soon as cells are adherent, you also might need to consider a different pore size. It is tough for a 40-50um cell to squeeze through 3/5um pores. 8um might be needed.
I hope that helps.
Yvonne
More
VOTE
Yvonne Baumer
Hi Yvonne, your suggestions for macrophage invasion are quite relevant. Can you share a protocol that works? i am just starting out with macrophage biology and any tip would be helpful. Thank you.
Yvonne Baumer
Hi Yvonne, your suggestions for macrophage invasion are quite relevant. Can you share a protocol that works? i am just starting out with macrophage biology and any tip would be helpful. Thank you.
More
VOTE
Hello Sehar,
THP-1s as you mentioned are non-adherent. I assume you checked the medium in the lower chamber for cells. Is that correct?
As soon as you have adherent cells you need to check the lower side of your membrane. Adherent cells will migrate through the pores but not deattach, meaning they are just on the other (bottom side) of your filter. For this you need to swipe the top inside with a cotton tip, fix the filter, label nuclei with DAPI and image the bottom of your filter.
THPs might not like being differentiated on a filter, so, you might have an additional factor. And as soon as cells are adherent, you also might need to consider a different pore size. It is tough for a 40-50um cell to squeeze through 3/5um pores. 8um might be needed.
I hope that helps.
Yvonne
Hello Sehar,
THP-1s as you mentioned are non-adherent. I assume you checked the medium in the lower chamber for cells. Is that correct?
As soon as you have adherent cells you need to check the lower side of your membrane. Adherent cells will migrate through the pores but not deattach, meaning they are just on the other (bottom side) of your filter. For this you need to swipe the top inside with a cotton tip, fix the filter, label nuclei with DAPI and image the bottom of your filter.
THPs might not like being differentiated on a filter, so, you might have an additional factor. And as soon as cells are adherent, you also might need to consider a different pore size. It is tough for a 40-50um cell to squeeze through 3/5um pores. 8um might be needed.
I hope that helps.
Yvonne
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VOTE
Ruchi Kakar - Please use the protocol described in this paper: https://www.jimmunol.org/content/199/10/3604.long#sec-2
That worked very well in our hands. Just write if you have any questions. Good luck.
Ruchi Kakar - Please use the protocol described in this paper: https://www.jimmunol.org/content/199/10/3604.long#sec-2
That worked very well in our hands. Just write if you have any questions. Good luck.
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Yvonne Baumer
Thank you so much for providing with the protocol :).
Yvonne Baumer
Thank you so much for providing with the protocol :).
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