I apologize for the late answer. Thank you Dr. Saluja and Dr. Lacy for your response. 1. As Dr. Saluja states, I am using mouse peritoneal mast cell lavage (PLMC) to acquire my primary cell line.This cells are supplemented with IMEM with FBS 10%, amino acids and antibiotics, etc. plus also IL-3 (5 ng/ml) and SCF (10 ng/ml) in 100 mm culture dishes for 2-3 weeks. Here is a recent reference of a protocol for isolation of PLMC or PMC: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918956/ 2. To answer Dr. Lacys question, I usually passage the cell cultures 1 or 2 times. Probably I have never done more passages than that. 3. To answer some questions about the protocol. We do usually include a positive control with phorbol 12-myristate (PMA) and ionomycin (calcium ionophore) and also protocol requires using lysates for calculating the amount of release (another way of depicting if my substrate is working). Again I don not know if p-NAG (4-Nitrophenyl N-acetyl-β- D -glucosaminide) is not a very sensitive substrate but my difference between negative control and positive control usually is around 60% secretion. The protocol calculates the fractional release of B-hex base on the lysate and supernatant. The real issue come with IgE sensitization, variability is a factor and as Dr, Lacy states I could change to a different substrate in order to increase sensitivity or perhaps change something in my cells to make them secrete more. As I stated in my original post, this cells used to secrete a lot and literature reviews them as cells that secrete greater quantities than BMMCs. Here is the protocol I am following exactly, it was published by Dr. Radinger recently: https://www.ncbi.nlm.nih.gov/pubmed/?term=Assay+of+Mast+Cell+Mediators+R%C3%A5dinger%2C+Madeleine+(et+al.) I appreciate a lot your answers and suggestions. If you have anything to add please do not hesitate to post.
I apologize for the late answer. Thank you Dr. Saluja and Dr. Lacy for your response. 1. As Dr. Saluja states, I am using mouse peritoneal mast cell lavage (PLMC) to acquire my primary cell line.This cells are supplemented with IMEM with FBS 10%, amino acids and antibiotics, etc. plus also IL-3 (5 ng/ml) and SCF (10 ng/ml) in 100 mm culture dishes for 2-3 weeks. Here is a recent reference of a protocol for isolation of PLMC or PMC: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918956/ 2. To answer Dr. Lacys question, I usually passage the cell cultures 1 or 2 times. Probably I have never done more passages than that. 3. To answer some questions about the protocol. We do usually include a positive control with phorbol 12-myristate (PMA) and ionomycin (calcium ionophore) and also protocol requires using lysates for calculating the amount of release (another way of depicting if my substrate is working). Again I don not know if p-NAG (4-Nitrophenyl N-acetyl-β- D -glucosaminide) is not a very sensitive substrate but my difference between negative control and positive control usually is around 60% secretion. The protocol calculates the fractional release of B-hex base on the lysate and supernatant. The real issue come with IgE sensitization, variability is a factor and as Dr, Lacy states I could change to a different substrate in order to increase sensitivity or perhaps change something in my cells to make them secrete more. As I stated in my original post, this cells used to secrete a lot and literature reviews them as cells that secrete greater quantities than BMMCs. Here is the protocol I am following exactly, it was published by Dr. Radinger recently: https://www.ncbi.nlm.nih.gov/pubmed/?term=Assay+of+Mast+Cell+Mediators+R%C3%A5dinger%2C+Madeleine+(et+al.) I appreciate a lot your answers and suggestions. If you have anything to add please do not hesitate to post.
I agree with Paige Lacy , number of passaging really matters , However as per my experience PLMCs show better degranulation as compared to RBL-2H3> I would prefer PMA/ ionomycin over IgE receptor cross linking to get better degranulation
I agree with Paige Lacy , number of passaging really matters , However as per my experience PLMCs show better degranulation as compared to RBL-2H3> I would prefer PMA/ ionomycin over IgE receptor cross linking to get better degranulation
How many passages have you subjected your mast cell line to? Passage number really impacts the degranulation responses. Cell lines generally lose their exocytotic capacity after a certain number of passages. I haven't used PLMCs but I know from RBL-2H3 cells that they lose their degranulation responses to cross-linked IgE after about 5-10 passages. You have to go back to the original stock to get the stronger degranulation responses.
How many passages have you subjected your mast cell line to? Passage number really impacts the degranulation responses. Cell lines generally lose their exocytotic capacity after a certain number of passages. I haven't used PLMCs but I know from RBL-2H3 cells that they lose their degranulation responses to cross-linked IgE after about 5-10 passages. You have to go back to the original stock to get the stronger degranulation responses.
Sorry, I was confused by the abbreviation as usually we refer to peritoneal mast cells as PMCs. In that case, the issue is very likely that the substrate is no longer effective for this assay, and colorimetry-based assays are not as sensitive as fluorescence-based assays for beta-hexosaminidase anyway. We get much better results using a fluorescence-based assay for beta-hexosaminidase, as attached. Your cell numbers per well should be fine. Using DNP-IgE is often variable in its effects, so you should include a positive control in each of your assays just to check that your substrate is working OK. The best positive controls are 48/80, calcium ionophores like A23187 or ionomycin, at around 5-10 uM. You should also include a sample of lysed cells to check that the substrate is working in your assay - cells should be lysed with 0.1% Triton X-100 or CHAPS. Hope this helps.
Sorry, I was confused by the abbreviation as usually we refer to peritoneal mast cells as PMCs. In that case, the issue is very likely that the substrate is no longer effective for this assay, and colorimetry-based assays are not as sensitive as fluorescence-based assays for beta-hexosaminidase anyway. We get much better results using a fluorescence-based assay for beta-hexosaminidase, as attached. Your cell numbers per well should be fine. Using DNP-IgE is often variable in its effects, so you should include a positive control in each of your assays just to check that your substrate is working OK. The best positive controls are 48/80, calcium ionophores like A23187 or ionomycin, at around 5-10 uM. You should also include a sample of lysed cells to check that the substrate is working in your assay - cells should be lysed with 0.1% Triton X-100 or CHAPS. Hope this helps.
Sorry, I was confused by the abbreviation as usually we refer to peritoneal mast cells as PMCs. In that case, the issue is very likely that the substrate is no longer effective for this assay, and colorimetry-based assays are not as sensitive as fluorescence-based assays for beta-hexosaminidase anyway. We get much better results using a fluorescence-based assay for beta-hexosaminidase, as attached. Your cell numbers per well should be fine. Using DNP-IgE is often variable in its effects, so you should include a positive control in each of your assays just to check that your substrate is working OK. The best positive controls are 48/80, calcium ionophores like A23187 or ionomycin, at around 5-10 uM. You should also include a sample of lysed cells to check that the substrate is working in your assay - cells should be lysed with 0.1% Triton X-100 or CHAPS. Hope this helps.
Sorry, I was confused by the abbreviation as usually we refer to peritoneal mast cells as PMCs. In that case, the issue is very likely that the substrate is no longer effective for this assay, and colorimetry-based assays are not as sensitive as fluorescence-based assays for beta-hexosaminidase anyway. We get much better results using a fluorescence-based assay for beta-hexosaminidase, as attached. Your cell numbers per well should be fine. Using DNP-IgE is often variable in its effects, so you should include a positive control in each of your assays just to check that your substrate is working OK. The best positive controls are 48/80, calcium ionophores like A23187 or ionomycin, at around 5-10 uM. You should also include a sample of lysed cells to check that the substrate is working in your assay - cells should be lysed with 0.1% Triton X-100 or CHAPS. Hope this helps.
The stop buffer used for termination of the reaction is just 0.2 M Tris base with no adjustment in the pH. This increases the pH of the reaction which effectively terminates the activity of hexosaminidase (optimal pH is 7.4 for this enzyme).
The stop buffer used for termination of the reaction is just 0.2 M Tris base with no adjustment in the pH. This increases the pH of the reaction which effectively terminates the activity of hexosaminidase (optimal pH is 7.4 for this enzyme).
@ Paige Lacy Hi Paige Lacy, Thanks for your Hexosaminidase assay fluorescent protocol. Could you let me know some details? 1. What's the pH of your tris-base buffer? 2. How do you make the 0.2M citrate buffer ? Thanks a lot for your help.
@ Paige Lacy Hi Paige Lacy, Thanks for your Hexosaminidase assay fluorescent protocol. Could you let me know some details? 1. What's the pH of your tris-base buffer? 2. How do you make the 0.2M citrate buffer ? Thanks a lot for your help.
is it mice or human mast cells , i think it is peritoneal mast cells ( mice)
If it is mice mast cell, you can follow my protocol
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043547
is it mice or human mast cells , i think it is peritoneal mast cells ( mice)
If it is mice mast cell, you can follow my protocol
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043547
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VOTE
I apologize for the late answer. Thank you Dr. Saluja and Dr. Lacy for your response.
1. As Dr. Saluja states, I am using mouse peritoneal mast cell lavage (PLMC) to acquire my primary cell line.This cells are supplemented with IMEM with FBS 10%, amino acids and antibiotics, etc. plus also IL-3 (5 ng/ml) and SCF (10 ng/ml) in 100 mm culture dishes for 2-3 weeks. Here is a recent reference of a protocol for isolation of PLMC or PMC:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918956/
2. To answer Dr. Lacys question, I usually passage the cell cultures 1 or 2 times. Probably I have never done more passages than that.
3. To answer some questions about the protocol. We do usually include a positive control with phorbol 12-myristate (PMA) and ionomycin (calcium ionophore) and also protocol requires using lysates for calculating the amount of release (another way of depicting if my substrate is working). Again I don not know if p-NAG (4-Nitrophenyl N-acetyl-β- D -glucosaminide) is not a very sensitive substrate but my difference between negative control and positive control usually is around 60% secretion. The protocol calculates the fractional release of B-hex base on the lysate and supernatant.
The real issue come with IgE sensitization, variability is a factor and as Dr, Lacy states I could change to a different substrate in order to increase sensitivity or perhaps change something in my cells to make them secrete more. As I stated in my original post, this cells used to secrete a lot and literature reviews them as cells that secrete greater quantities than BMMCs.
Here is the protocol I am following exactly, it was published by Dr. Radinger recently:
https://www.ncbi.nlm.nih.gov/pubmed/?term=Assay+of+Mast+Cell+Mediators+R%C3%A5dinger%2C+Madeleine+(et+al.)
I appreciate a lot your answers and suggestions. If you have anything to add please do not hesitate to post.
I apologize for the late answer. Thank you Dr. Saluja and Dr. Lacy for your response.
1. As Dr. Saluja states, I am using mouse peritoneal mast cell lavage (PLMC) to acquire my primary cell line.This cells are supplemented with IMEM with FBS 10%, amino acids and antibiotics, etc. plus also IL-3 (5 ng/ml) and SCF (10 ng/ml) in 100 mm culture dishes for 2-3 weeks. Here is a recent reference of a protocol for isolation of PLMC or PMC:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918956/
2. To answer Dr. Lacys question, I usually passage the cell cultures 1 or 2 times. Probably I have never done more passages than that.
3. To answer some questions about the protocol. We do usually include a positive control with phorbol 12-myristate (PMA) and ionomycin (calcium ionophore) and also protocol requires using lysates for calculating the amount of release (another way of depicting if my substrate is working). Again I don not know if p-NAG (4-Nitrophenyl N-acetyl-β- D -glucosaminide) is not a very sensitive substrate but my difference between negative control and positive control usually is around 60% secretion. The protocol calculates the fractional release of B-hex base on the lysate and supernatant.
The real issue come with IgE sensitization, variability is a factor and as Dr, Lacy states I could change to a different substrate in order to increase sensitivity or perhaps change something in my cells to make them secrete more. As I stated in my original post, this cells used to secrete a lot and literature reviews them as cells that secrete greater quantities than BMMCs.
Here is the protocol I am following exactly, it was published by Dr. Radinger recently:
https://www.ncbi.nlm.nih.gov/pubmed/?term=Assay+of+Mast+Cell+Mediators+R%C3%A5dinger%2C+Madeleine+(et+al.)
I appreciate a lot your answers and suggestions. If you have anything to add please do not hesitate to post.
More
VOTE
I agree with Paige Lacy , number of passaging really matters , However as per my experience PLMCs show better degranulation as compared to RBL-2H3> I would prefer PMA/ ionomycin over IgE receptor cross linking to get better degranulation
I agree with Paige Lacy , number of passaging really matters , However as per my experience PLMCs show better degranulation as compared to RBL-2H3> I would prefer PMA/ ionomycin over IgE receptor cross linking to get better degranulation
More
VOTE
I have never heard of PLMC cells as a mast cell line. Would you be able to provide a literature reference for these cells?
I have never heard of PLMC cells as a mast cell line. Would you be able to provide a literature reference for these cells?
More
VOTE
How many passages have you subjected your mast cell line to? Passage number really impacts the degranulation responses. Cell lines generally lose their exocytotic capacity after a certain number of passages. I haven't used PLMCs but I know from RBL-2H3 cells that they lose their degranulation responses to cross-linked IgE after about 5-10 passages. You have to go back to the original stock to get the stronger degranulation responses.
How many passages have you subjected your mast cell line to? Passage number really impacts the degranulation responses. Cell lines generally lose their exocytotic capacity after a certain number of passages. I haven't used PLMCs but I know from RBL-2H3 cells that they lose their degranulation responses to cross-linked IgE after about 5-10 passages. You have to go back to the original stock to get the stronger degranulation responses.
More
VOTE
Sorry, I was confused by the abbreviation as usually we refer to peritoneal mast cells as PMCs. In that case, the issue is very likely that the substrate is no longer effective for this assay, and colorimetry-based assays are not as sensitive as fluorescence-based assays for beta-hexosaminidase anyway. We get much better results using a fluorescence-based assay for beta-hexosaminidase, as attached. Your cell numbers per well should be fine. Using DNP-IgE is often variable in its effects, so you should include a positive control in each of your assays just to check that your substrate is working OK. The best positive controls are 48/80, calcium ionophores like A23187 or ionomycin, at around 5-10 uM. You should also include a sample of lysed cells to check that the substrate is working in your assay - cells should be lysed with 0.1% Triton X-100 or CHAPS. Hope this helps.
Sorry, I was confused by the abbreviation as usually we refer to peritoneal mast cells as PMCs. In that case, the issue is very likely that the substrate is no longer effective for this assay, and colorimetry-based assays are not as sensitive as fluorescence-based assays for beta-hexosaminidase anyway. We get much better results using a fluorescence-based assay for beta-hexosaminidase, as attached. Your cell numbers per well should be fine. Using DNP-IgE is often variable in its effects, so you should include a positive control in each of your assays just to check that your substrate is working OK. The best positive controls are 48/80, calcium ionophores like A23187 or ionomycin, at around 5-10 uM. You should also include a sample of lysed cells to check that the substrate is working in your assay - cells should be lysed with 0.1% Triton X-100 or CHAPS. Hope this helps.
More
VOTE
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918956/
Paige Lacy - Here is the protocol for peritoneal mast cell preparation, these are not cell line
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918956/
Paige Lacy - Here is the protocol for peritoneal mast cell preparation, these are not cell line
More
VOTE
Sorry, I was confused by the abbreviation as usually we refer to peritoneal mast cells as PMCs. In that case, the issue is very likely that the substrate is no longer effective for this assay, and colorimetry-based assays are not as sensitive as fluorescence-based assays for beta-hexosaminidase anyway. We get much better results using a fluorescence-based assay for beta-hexosaminidase, as attached. Your cell numbers per well should be fine. Using DNP-IgE is often variable in its effects, so you should include a positive control in each of your assays just to check that your substrate is working OK. The best positive controls are 48/80, calcium ionophores like A23187 or ionomycin, at around 5-10 uM. You should also include a sample of lysed cells to check that the substrate is working in your assay - cells should be lysed with 0.1% Triton X-100 or CHAPS. Hope this helps.
Sorry, I was confused by the abbreviation as usually we refer to peritoneal mast cells as PMCs. In that case, the issue is very likely that the substrate is no longer effective for this assay, and colorimetry-based assays are not as sensitive as fluorescence-based assays for beta-hexosaminidase anyway. We get much better results using a fluorescence-based assay for beta-hexosaminidase, as attached. Your cell numbers per well should be fine. Using DNP-IgE is often variable in its effects, so you should include a positive control in each of your assays just to check that your substrate is working OK. The best positive controls are 48/80, calcium ionophores like A23187 or ionomycin, at around 5-10 uM. You should also include a sample of lysed cells to check that the substrate is working in your assay - cells should be lysed with 0.1% Triton X-100 or CHAPS. Hope this helps.
More
VOTE
The stop buffer used for termination of the reaction is just 0.2 M Tris base with no adjustment in the pH. This increases the pH of the reaction which effectively terminates the activity of hexosaminidase (optimal pH is 7.4 for this enzyme).
The stop buffer used for termination of the reaction is just 0.2 M Tris base with no adjustment in the pH. This increases the pH of the reaction which effectively terminates the activity of hexosaminidase (optimal pH is 7.4 for this enzyme).
More
VOTE
@ Paige Lacy
Hi Paige Lacy, Thanks for your Hexosaminidase assay fluorescent protocol. Could you let me know some details? 1. What's the pH of your tris-base buffer?
2. How do you make the 0.2M citrate buffer ? Thanks a lot for your help.
@ Paige Lacy
Hi Paige Lacy, Thanks for your Hexosaminidase assay fluorescent protocol. Could you let me know some details? 1. What's the pH of your tris-base buffer?
2. How do you make the 0.2M citrate buffer ? Thanks a lot for your help.
More
VOTE