Standardizacija protokola meritev L-laktata v posamezni celici 3T3-L1

Maver, Anja (author), Zorec, Robert (mentor)

L-laktat je pomemben metabolit, ki nastaja pri anaerobni in aerobni glikolizi ter ima ključno vlogo pri celični presnovi in signalizaciji. V celicah aktivira laktatne receptorje, med katere spada tudi GPR81; slednji je prisoten v astrocitih v možganskem tkivu. Raziskava, pri kateri so proučevali astrocite z izbitim genom za GPR81, je pokazala, da so v astrocitih poleg GPR81 prisotni tudi drugi, še neidentificirani laktatni receptorji. Za identifikacijo le-teh so potrebne nadaljnje študije, pri katerih bi preko nanosenzorjev FRET s fluorescenčno mikroskopijo merili spremembe citosolnega L-laktata v celicah v mirovanju ali po stimulaciji z zunajceličnim L-laktatom oz. ligandi Smart. Meritve s fluorescenčno mikroskopijo vključujejo osvetlitve celic, pri čemer je pomembno, kolikšna je intenziteta svetlobe ter kolikšni so ekspozicijski časi ekscitacije fluorescence. Predolga ekspozicija celic na svetlobi ter previsoka intenziteta svetlobe povzročata fototoksične učinke na celice, kar se pozna pri kvaliteti eksperimentov. Postopek meritve citosolnega L-laktata je zato potrebno standardizirati in določiti, ali ekspozicijski čas ekscitacije fluorescence, ki ga uporabljamo pri eksperimentih, signifikantno vpliva na odzive celic po stimulaciji z 20 mM zunajceličnim L-laktatom oz. ligandom Smart009 ali Smart075 v koncentracijah 1000 µM in 500 µM. Da bi določili, ali ta odvisnost obstaja, smo celice 3T3-L1 WT in 3T3-L1 KO19 transficirali z nanosenzorjem FRET Laconic, ki detektira L-laktat v fiziološkem obsegu, ter jih stimulirali z zunajceličnim L-laktatom oz. ligandoma Smart. Pri tem smo s fluorescenčnim mikroskopom zajemali slike pri različnih ekspozicijskih časih ekscitacije fluorescence ter ustvarili posnetke celic v realnem času. Iz analize posnetkov smo dobili spremembe v signalu FRET, ki kažejo spremenjeno citosolno koncentracijo L-laktata oz. t. i. odziv celice. Podatke o ekspozicijskih časih smo za posamezno celico združili s podatki o spremenjenem signalu FRET in združene podatke nato analizirali s testom linearne regresije. Pri statistični analizi so nas zanimali korelacijski faktor r, vrednost P in determinacijski koeficient r2. Rezultati kažejo, da spremembe v signalu FRET niso v nobenem primeru stimulacije statistično signifikantno odvisne od ekspozicijskega časa.

• Language: Slovenian
• Keywords: 3T3-L1, laktatni receptor, L-laktat, fototoksičnost
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2020
• PID: 20.500.12556/RUL-118760
• COBISS.SI-ID: 27233283
• Publication date in RUL: 31.08.2020

Secondary language

• Language: English
• Title: Standardization of protocol for measurements of L-lactate in single 3T3-L1 cell

Abstract

L-lactate is a cell metabolite, produced in the process of anaerobic and aerobic glycolysis and plays an important role as a fuel and a signal. It activates lactate receptors, including GPR81, which is present in astrocytes. A study with GPR81-knockout astrocytes showed, that there are other, yet unidentified, lactate receptors in addition to GPR81. Further studies are needed to identify these lactate receptors, which include studies of measuring changes in cytosolic L-lactate at rest or after stimulation with extracellular L-lactate or Smart ligands, agonists elevating cytosolic L-lactate. To measure cytosolic L-lactate we used Förster Resonance Energy Tranfer (FRET) nanosensors using fluorescence microscopy. Measurements by fluorescence microscopy involves illuminating the cells; in these experiments the intensity of light as well as the exposure time are crucial for the quality of the results. Long exposure time or too intense light can cause phototoxic effects on cells, which is evident in the quality of the experiments. The cytosolic L-lactate measurement protocol should therefore be standardized and it is necessary to determine whether the fluorescence excitation exposure time used in the experiments significantly affects cell responses after stimulation with 20 mM extracellular L-lactate and ligands Smart009 or Smart075 at concentrations of 1000 μM and 500 μM. To examine this particular correlation, we performed transfection of 3T3-L1 WT and 3T3-L1 KO19 cells with the FRET nanosensor Laconic, which detects L-lactate in physiological range, and stimulated cells with extracellular L-lactate or Smart ligands. The fluorescence microscope was used to capture images in real time at different exposure times of fluorescence excitation. The results revealed that changes in the FRET signal, which report changes in cytosolic concentration of L-lactate (i.e. cell response) are independent of the exposure time. This conclusion is based on the statistical analysis, where the correlation factor r, the P value and the coefficient of determination r2 were obtained. The results show that the changes in the FRET signal are in no case statistically significantly dependent on the exposure time.

• Keywords: 3T3-L1, lactate receptor, L-lactate, phototoxicity