Hi Imr,

Thank you for your question.  The first thing I want to point out is that you want to make sure your cells are adhered to the bottom of the well.  I am not sure which type of instrument you have, but below are our protocols for adhering non-adherent cells via Cell-Tak:

Immobilization of Non-Adherent Cells with Cell-Tak for Assay on the Agilent Seahorse XFe/XF24 Analyzer

Immobilization of Non-Adherent Cells with Cell-Tak for Assay on the Agilent Seahorse XFe/XF96 or XFp Analyzer

You do want them to be as pure as possible because any other cells that are seeded in the plate will contribute to your rates.  We do not have an official recommendation as far as purifying methods, but you can use our Cell Reference Database to see what other users have done to purify.  You can change the “Cell Type” drop-down to monocytes – there are many different papers listed that should be helpful!

Here are some papers I was able to find on there that I thought you might find interesting:

1.  Rewiring monocyte glucose metabolism via C-type lectin signaling protects against disseminated candidiasis

2.  Commitment to glycolysis sustains survival of NO-producing inflammatory dendritic cells 

3.  Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood 

4.  Aging impairs mitochondrial respiratory capacity in classical monocytes

5.  Classical monocytes maintain ex vivo glycolytic metabolism and early but not later inflammatory responses in older adults

6.  Candida albicans β-Glucan Differentiates Human Monocytes Into a Specific Subset of Macrophages

7.  Monocytes from Older Adults Maintain Capacity for Metabolic Compensation during Glucose Deprivation and Lipopolysaccharide Stimulation

8.  The Bioenergetic Health Index: a new concept in mitochondrial translational research

9.  Blood-Based Bioenergetic Profiling Reflects Differences in Brain Bioenergetics and Metabolism

10.  Decreased Bioenergetic Health Index in monocytes isolated from the pericardial fluid and blood of post-operative cardiac surgery patients

11.  Assessing Functional Metabolism Using Cells Sourced from Blood Real-time, Kinetic Measurements Link Mitochondrial Function to Disease Diagnosis and Progression

Additionally, here is a Jove Video specific to leukocytes  that you may find helpful.

Please let me know if you have any other questions after taking a look at these documents/links.

Thanks!

Courtney

Hi Imr,

Thank you for your question.  The first thing I want to point out is that you want to make sure your cells are adhered to the bottom of the well.  I am not sure which type of instrument you have, but below are our protocols for adhering non-adherent cells via Cell-Tak:

Immobilization of Non-Adherent Cells with Cell-Tak for Assay on the Agilent Seahorse XFe/XF24 Analyzer

Immobilization of Non-Adherent Cells with Cell-Tak for Assay on the Agilent Seahorse XFe/XF96 or XFp Analyzer

You do want them to be as pure as possible because any other cells that are seeded in the plate will contribute to your rates.  We do not have an official recommendation as far as purifying methods, but you can use our Cell Reference Database to see what other users have done to purify.  You can change the “Cell Type” drop-down to monocytes – there are many different papers listed that should be helpful!

Here are some papers I was able to find on there that I thought you might find interesting:

1.  Rewiring monocyte glucose metabolism via C-type lectin signaling protects against disseminated candidiasis

2.  Commitment to glycolysis sustains survival of NO-producing inflammatory dendritic cells 

3.  Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood 

4.  Aging impairs mitochondrial respiratory capacity in classical monocytes

5.  Classical monocytes maintain ex vivo glycolytic metabolism and early but not later inflammatory responses in older adults

6.  Candida albicans β-Glucan Differentiates Human Monocytes Into a Specific Subset of Macrophages

7.  Monocytes from Older Adults Maintain Capacity for Metabolic Compensation during Glucose Deprivation and Lipopolysaccharide Stimulation

8.  The Bioenergetic Health Index: a new concept in mitochondrial translational research

9.  Blood-Based Bioenergetic Profiling Reflects Differences in Brain Bioenergetics and Metabolism

10.  Decreased Bioenergetic Health Index in monocytes isolated from the pericardial fluid and blood of post-operative cardiac surgery patients

11.  Assessing Functional Metabolism Using Cells Sourced from Blood Real-time, Kinetic Measurements Link Mitochondrial Function to Disease Diagnosis and Progression

Additionally, here is a Jove Video specific to leukocytes  that you may find helpful.

Please let me know if you have any other questions after taking a look at these documents/links.

Thanks!

Courtney

Courtney Watts said:

Immobilization of Non-Adherent Cells with Cell-Tak for Assay on the Agilent Seahorse XFe/XF96 or XFp Analyzer

I have been following this protocol for cell attachment but the cells still couldn't attached. In the technical note, sodium hydroxide was mentioned in the Materials, but the use of sodium hydroxide was not mentioned in the protocol steps. The protocol in Cell-Tak is a little different. I am a bit confused. Please help. 

Hi there, 

Thank you for contacting Cell Analysis Technical Support.  The protocol you mentioned does reference the manufacturer of Cell Tak Basic Protocol (see screenshot below).  This is where the sodium hydroxide is used to neutralize the Cell Tak for the adhesive function to be activated.  Have you gone to that referenced protocol as well?  You will see it there.  Please let me know if this helps or if you have any additional questions once you take a look.

Thank you,

Courtney