Propidium Iodide is one of the most common and recommended dyes to use to quantitatively assess DNA content by flow cytometry (Darzynkiewicz et al 2001). Does Kratom Have Health Benefits the dose response and temporal effects of treatment were examined in this assay in order to maximally evaluate the effect on the cell cycle. Cell cycle analysis was initially performed using HEK 293 cells and the DNA profile was determined manually using the Cellquest Pro software (Fig.
Genome maintenance mechanisms for preventing cancer. Nature 411: 366-374. P53 mutations in human cancers. Science 253: 49-53. Sofuni T (1999).
For MIT treated cells changes of the four populations were not as drastic as MSE treated cells. Q3 and Q4 indicating increased of apoptotic and necrotic cells. For MCL-5 cells (Fig 5. The majority of the cells were evidently located in the Q3 and Q4 indicating the necrotic and late stage of apoptotic populations. This finding supports the cytological examinations previously noted where the cells were predominantly necrotic and in the late stage of apoptosis. Control 1 10 50 100 250 91. Q2 (%) 3.
Relative suspension growth (RSG) 91. Y Y Y Y Y Y Y Y Y Y Y Conc. Summary table of MLA result for MSE in the i) presence of S9 and ii) in the absence of S9. S9 treatment Treatment groups Negative control MSE 0 0 0 40 30 20 Positive control (MMS) Mean Control MF 75. Negative Negative Negative Negative Negative Negative Positive Conc.
To further confirm the outcome seen in the Alamar blue assay experiments (Fig. DED and ATZ was employed. From the result (Fig. DED a CYP 2A6 inhibitor also
gave some protection against MSE and MIT toxicity but was not effective as ATZ.
Values are the mean of quadruplet cultures of MSE experiment and duplicate cultures of MIT experiment. Bars are SEM. ANOVA with Dunnet post test. IC50 values (Inhibition concentration that caused 50% cell death) of 24 hr treatment with MSE and MIT treated cell lines. The values were interpolated from percentage dead cells curves obtained from the Trypan blue exclusion experiments. MIT (Molar) 7.
Therefore an alternative assay (Trypan blue exclusion) was used to examine the effect of higher concentrations of MSE on cell toxicity. kratom tea health benefits Effect of MSE on cytotoxicity (A) and proliferation (B) of HepG2 cells after 24 hr of treatment. The enzymatic reaction (LDH activity) was determined by fluorescence with an excitation wavelength of 560 nm and emission wavelength of 590 nm. Values are means of triplicates. Bars are standard error of the mean (SEM). To assess the effect of MSE on cell proliferation and viability the Trypan Blue exclusion assay was kratom mitragyna speciosa effects performed. This assay could be used with much higher concentrations of MSE and showed dose and time-dependency in cell proliferation and viability.
As anticipated toxicity effects seen at high doses Does Kratom Have Health Benefits suggested apoptotic morphology with evidence of chromatin condensation which was predominantly seen in SH-SY5Y cells. Nuclear alterations are key in many descriptions of apoptosis. The severity of MSE insult in the SH-SY5Y cell line was obvious at the highest dose tested as there were very few cells present on the slide and all of them showed apoptotic morphology.
The increase of subG1 population was also prominent at these two highest doses. DNA replication process occurring (increased S phase cells). This finding was found to be in contrast to the previous MCL-5 results (Fig. The control cells also show a similar DNA profile as the treated cells at the same time point. The S phase population remains active until the 8 hr treatment period. M phase cells.
Bars are the mean kratom make you high of three experiments with SEM. P53 levels of MSE treated SH-SY5Y cells at different time points (6 12 24 and 48 hr). P53 levels of MIT treated SH-SY5Y cells after 24 hr treatment. P53 levels of MIT treated SH-SY5Y cells at different time points (6 12 24 and 48 hr).
Over 25 alkaloids have been isolated from kratom. The most abundant alkaloids consist of three indoles and two oxindoles. The three indoles are mitragynine paynanthine and speciogynine; the first two of which appear to be unique to this species. The two oxindoles are mitraphylline and speciofoline.
The trypan blue assay and clonogenicity assay were employed as described in chapter 2 section 2. MSE and MIT are discussed as follows: Effects of naloxone on MSE and MIT treated cells: Fig. Naloxone also appears to successfully inhibit the MIT toxicity (Fig.
Cytological examination of MSE treated cells Cytological examinations
were carried out using SH-SY5Y HEK 293 and MCL-5 cells. Staining of these treated cells were performed using Wright-Giemsa or Rapi-Diff staining as they offered a quick and a general purpose stain. HEK 293 MCL-5 and SH-SY5Y cells (2 x 105) were cultured in 25 cm2 flasks containing 6 ml media and were acclimatised Does kratom world seed supply Kratom Have Health Benefits Does Kratom Have Health Benefits overnight for HEK 293 and SHSY5Y cells and 2 hr for MCL-5 cells prior to treatment with various concentration of MSE. C (5% CO2) for the designated time period. The adherent cells (HEK 293 and SH-SY5Y cells) were harvested trypsinised and centrifuged as per routine procedures described in chapter 2 sections 2. After this incubation the cells were harvested as previously described (section 2.