Tim Sakogawa
Cell Culture Reading Assignment
A) Miao, J.Y.; Shen, S.L.; Wang, H.Y.; Wang, S.Q.; Wu, Q.H.; Zhang, Y.R.; Zhao, B.X. Novel Pyrazoline-Based Fluorescent Probe for Detecting Glutathione and its Application in Cells. Biosens. Bioelectron. 2014, 55, 386-390.
B) The authors of this paper were able to develop a new highly sensitive fluorescent probe with low toxicity. The probe is based on a pyrazoline unit and detects GSH (glutathione) in buffered EtOH:PBS with a ratio of 3:7, pH 7.4. This probe has a detection limit of 8.2 * 10^-8 M which is extremely good. This means that the probe is capable of detecting the presence of GSH down to a molarity of 8.2 * 10^-8 M.
C) In this paper, the researchers used HeLa cells. The paper doesn’t give an exact reason as to why they used HeLa cells, but I’m assuming it’s because these cells are very easy to grow and they also grow very fast. I’ve noticed that out of all the cells that we’re maintaining in lab, the HeLa’s seem to be the easiest and fastest growing cells. Time may have been limited during their development of the probe, so choosing a rapidly growing line such as HeLas would have been a very favorable choice.
D) Cytotoxicity Assay: They plated 4x10^4 cells/well in a 96 well plate and fixed them with varying concentrations of the probe. They then fixed the cells with 4% TCA for 1h at 4 Degrees C and washed with de-ionized water. They then added 50 uL of SRB (sulforhodamine B) to each well, then added Tris-HCl and acetic acid solution to get rid of the SRB. Absorbance was the checked at 540nm.
Microscopic study with probe L: The authors cultured HeLa cells much like we do in class, using DMEM, incubating at 37 degrees C, and probably using hemocytometers for counting cells and making appropriate concentrations for their solutions. The HeLa cells were incubated with probe L similar to how the Geneticin was incubated in our CV-1 cells, and then they were checked using a fluorescent microscope much like we did.
E) In this paper the question being asked was: how can we make a highly sensitive fluorescent probe that is selective for the detection of GSH (glutathione).
F) When you were testing the selectivity of this probe L for GSH, you treated 10 uL of probe with 100 uL of analyte in PBS. You saw that this increased its fluorescence intensity by up to 83 fold. Why did you not try a different ratio of probe L to analyte such as 1:5 instead? Do you think this would have increased the intensity of the fluorescence, thereby making it easier to detect?
When you combined the probe L with analyte, it is stated that the analyte contains Cys, Hcy, GSH, Arg, Asp, Glu, Gly, His, Lys, Ser, Thr, Trp, Tyr, Val, K+, Ca2+, Na+, Mg2+, Zn2+, Fe3+, peroxide and glucose. What is the purpose of using this specific analyte when trying to test for fluorescence? Does it have to be this exact type of analyte or can we use another type of analyte with other substances