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Difference between revisions of "Microwell respirometry"

From Bioblast
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== OROBOROS comments on microplate respirometry ==
== OROBOROS comments on microplate respirometry ==
  '''In respirometry, high throughput is ''not equivalent to high output''.'''
  '''In respirometry, high throughput is ''not equivalent to high output''.'''
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== Respirometry with cryopreserved blood cells ==
== Respirometry with cryopreserved blood cells ==
* Successful application of cryopreserved blood cells is reported by [[DE_Ulm_Karabatsiakis A |Karabatsiakis and colleagues]] (2014) <ref> Karabatsiakis A, Bock C, Salinas-Manrique J, Kolassa S, Calzia E, Dietrich DE, Kolassa I-T (2014) Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression. Translational Psychiatry 4:e397. [[Karabatsiakis 2014 Translational Psychiatry |»Open Access«]] </ref> with O2k high-resolution respirometry based on
* Successful application of cryopreserved blood cells is reported by [[DE_Ulm_Karabatsiakis A |Karabatsiakis and colleagues]] (2014) <ref> Karabatsiakis A, Bock C, Salinas-Manrique J, Kolassa S, Calzia E, Dietrich DE, Kolassa I-T (2014) Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression. Translational Psychiatry 4:e397. [[Karabatsiakis 2014 Translational Psychiatry |»Open Access«]] </ref> with O2k high-resolution respirometry based on
::(1) functinal assays with blood cells in suspension;
::(1) functional assays with blood cells in suspension;
::(2) immediate measurement in the OROBOROS O2k after thawing upon cryopreservation.
::(2) immediate measurement in the OROBOROS O2k after thawing upon cryopreservation.


* In contrast, Keane et al (2015) <ref> Keane KN, Calton EK, Cruzat VF, Soares MJ, Newsholme P (2015) The impact of cryopreservation on human peripheral blood leukocyte bioenergetics. Clin Sci (Lond) 128:723–33. [[Keane 2015 Clin Sci (Lond) |»Open Access«]] </ref> found mitochondrial injuries after cryopreservation of blood cells. The authors state: ''The Seahorse XFe96 Flux analyzer was used according to manufacturer's instructions. .. In brief, cells were seeded into 96-well plates at a density of 3.5 x 10_5 cells/well and allowed to '''adhere overnight. .. then incubated for 60 min at 37 °C in an non-CO2 incubator, to allow the cells to become equilibrated with the new medium.''''' The reductions in [[ROUTINE respiration]], [[ETS capacity]] and coupling after 1 month cryopreservation may be related to
* In contrast, Keane et al (2015) <ref> Keane KN, Calton EK, Cruzat VF, Soares MJ, Newsholme P (2015) The impact of cryopreservation on human peripheral blood leukocyte bioenergetics. Clin Sci (Lond) 128:723–33. [[Keane 2015 Clin Sci (Lond) |»Open Access«]] </ref> found mitochondrial injuries after cryopreservation of blood cells. The authors state: ''The Seahorse XFe96 Flux analyzer was used according to manufacturer's instructions. .. In brief, cells were seeded into 96-well plates at a density of 3.5 x 10<sup>5</sup> cells/well and allowed to '''adhere overnight. .. then incubated for 60 min at 37 °C in an non-CO<sub>2</sub> incubator, to allow the cells to become equilibrated with the new medium.''''' The reductions in [[ROUTINE respiration]], [[ETS capacity]] and coupling after 1 month cryopreservation may be related to
::(1) measurement on blood cells adherent in microwells;
::(1) measurement on blood cells adherent in microwells;
::(2) delayed measurement following overnight stress condition.
::(2) delayed measurement following overnight plus 60 min stress condition.


'''References'''
'''References'''
<references/>
<references/>

Revision as of 12:30, 28 May 2015

OROBOROS comments on microplate respirometry

In respirometry, high throughput is not equivalent to high output.
» O2k versus multiwell respirometer


Are microwell cell cultures physiological?

Biologists go to great lengths to figure out just the right cocktail of chemicals on which to propagate the cells they want to study. Not too much nitrogen—just the right amount of sulfur—and so on. But then the whole mix is thrown into a flat-bottomed microwell where the newly sprouted cells bunch up and do not exactly look or behave like cells grown au naturel. Hoping to give lab-cultured cells the chance to more closely resemble their in vivo brethren, materials science researchers have designed a technique for growing cells in three dimensions on top of a cushion of air.


Respirometry with cryopreserved blood cells

(1) functional assays with blood cells in suspension;
(2) immediate measurement in the OROBOROS O2k after thawing upon cryopreservation.
  • In contrast, Keane et al (2015) [2] found mitochondrial injuries after cryopreservation of blood cells. The authors state: The Seahorse XFe96 Flux analyzer was used according to manufacturer's instructions. .. In brief, cells were seeded into 96-well plates at a density of 3.5 x 105 cells/well and allowed to adhere overnight. .. then incubated for 60 min at 37 °C in an non-CO2 incubator, to allow the cells to become equilibrated with the new medium. The reductions in ROUTINE respiration, ETS capacity and coupling after 1 month cryopreservation may be related to
(1) measurement on blood cells adherent in microwells;
(2) delayed measurement following overnight plus 60 min stress condition.

References

  1. Karabatsiakis A, Bock C, Salinas-Manrique J, Kolassa S, Calzia E, Dietrich DE, Kolassa I-T (2014) Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression. Translational Psychiatry 4:e397. »Open Access«
  2. Keane KN, Calton EK, Cruzat VF, Soares MJ, Newsholme P (2015) The impact of cryopreservation on human peripheral blood leukocyte bioenergetics. Clin Sci (Lond) 128:723–33. »Open Access«