- 2015 - Florian: To obtain several data from only one sample, protocols were prepared for the separation of various human blood cells. In order to obtain thrombocytes and leukocytes/monocytes (PBMCs) or thrombocytes, PBMCs and granulocytes, specially adapted separation methods were further developed from the literature and optimized for use in the O2k, with the main attention being placed on the use of the lowest possible total blood volume, in order to keep the physical burden on subjects as low as possible. Furthermore, the specific titration protocols for the individual blood cell types were optimized with regard to certain concentrations of titrations and with regard to the respiratory media used. Gained conclusions have been applied in a field study.
- Elisa Calabria sent an email with the protocols (PBMCs) which she used with Florian (and she is still using in Verona). Attached the protocol for intact and permeabilized PBMCs.
Doerrierc 15:38, 20 January 2016 (CET)
1. Development of separation protocols
The following steps were carried out to optimize the time required for the separation and cell viability:
Accomplished actions:
- Literary research on existing separation methods of thrombocytes, PBMCs, granulocytes
- Combination in the literature of existing methods to more complex separation protocols for varied human blood cell types
- Comparison of centrifugation forces diverging in the literature for the separation/washing of cells (increase of cell viability)
- Comparison of different washing media to optimize cell viability (PBS vs. RPMI 1640)
- Development of a method for reliable estimating of cells (v.a. thrombocytes) - still QUESTIONABLE!
- Experiments on the reproducibility of respirometric results
- Comparison and ongoing adaptation of diverse separation protocols to optimize cell viability and separation time
2. Progression of SUIT-protocols
Fundamentally, was based on titration protocols used by Eskils Elmer's research group (Univerity Lund)(Thrombocytes and PBMCs. Further research activities with granulocytes were dispensed with due to the unclean results of specific density centrifugation, of the very low respirometric activity or respectively apoptotic response to substrate titrations.
Accomplished actions:
- Adaptation of various titration concentrations recommended by Eskil Elmer's research group to current findings of Oroboros Instruments (Malate, Antimycin A)
- Experimental verification of final concentrations in Digitonin, Rotenone and Amplex Red in protocols for thrombocytes and PBMCs
- Adaptation of the protocols for the possible parallel use of fluorometric investigations using the LED-2 modules (Additional titrations of Amplex Red, Horseradish Peroxidase and H2O2)
- Comparison of varied respiratory media in intact cells (PBS vs. PBS glucose vs. RPMI 1640) or permeabilized cells (MiR05 vs. MiR05 Cr)
- Test application of Phorbol-12-myristate-13-acetate (PMA) in the O2k for the induction and measurement of the oxidative burst in thrombocytes and PBMCs
- Test application of octanoylcarnitines, a-glycerophosphates and S50 and implementation in the current SUIT-protocol
3. Application in an exemplary field study
For the exemplary application of prepared protocols, a field study (Comparison of respirometric values on Ultramarathon runners before/after participation in the competition) on permeabilized thrombozytes has been performed. This should illustrate the problem-free and simple application of these in a suboptimal environment without laboratory requirements questions in the areas of motion and sport science, or to illustrate possible problems that still arise.
Accomplished actions:
- Development and test application of a separation and titration protocol adapted to the study methodology
- Organizational actions for the frictionless running of the project(Subjects search, fixation of the examination site near the start/finish area of the marathon, detailed investigation during the surveys, to be investigated secondary parameters, scientific staff on the three survey days, organization for all necessary equipment)
- Analysis of the main parameters (respirometric data)
- Further analyzes of various secondary parameters (Analysis protein content, analysis citrate synthase)
- Statistical analyzes of the results (NOT COMPLETED)
- Summary of preliminary results for the participants or respectively sending data sheets of the personal test results
