Method for rapidly detecting soothing effect of cosmetics through mast cell degranulation state
A technology of mast cells and cosmetics, applied in the field of biomedicine, can solve the problems of not being very good, unable to give quantitative results, and unable to reflect the degranulation status of a single mast cell, so as to achieve the effect of inhibition
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Embodiment 1
[0060] Example 1. Comparison of Avidin and CD63 to detect mast cell degranulation effect
[0061] In this protocol, mast cell P815 was stimulated with C48 / 80 at 0 μg / mL and 100 μg / mL for 15 minutes, and mast cell degranulation was analyzed by Avidin-FTIC or CD63-FITC staining. C48 / 80 stimulation at 100 μg / mL significantly induced mast cell degranulation ( figure 1 ). CD63-FTIC can detect mast cell degranulation. However, Avidin-FITC recognized a higher proportion of degranulated mast cells than CD63-FITC ( figure 1 ).
Embodiment 2
[0062] Example 2. Establishment of C48 / 80 in vitro induced degranulation test conditions
[0063] The effects of 10 μg / mL and 100 μg / mL C48 / 80 on degranulation of P815 cells after 1 h of stimulation were tested in this protocol. By direct morphological observation, 10 μg / mL C48 / 80 significantly induced mast cell degranulation ( figure 2 ), while 100 μg / mL C48 / 80 resulted in degranulation of all mast cells. Therefore, 10 μg / mL C48 / 80 was selected as the in vitro stimulating concentration for subsequent experiments in this protocol.
Embodiment 3
[0064] Example 3. Establishment of sodium cromolate as a positive control for inhibition of mast cell degranulation
[0065] 1) Administration
[0066] Add 50 μL of resuspended P815 cells (or human mast cells LAD2 or HMC-1) to a 1.2 mL flow cytometry tube, add serum-free high-glucose DMEM medium to the negative control group, and add 50 μL of 1 mg / mL sodium cromoglycate to the positive control group of serum-free high-glucose DMEM medium, with a total volume of 100 μL per well. After dosing, cells were placed in an incubator (37°C, 5% CO 2 , 95% RH) for 2 hours.
[0067] 2) Degranulation induction
[0068] Quickly add 0 μL, 1 μL, and 2 μL of C48 / 80 solution (1 mg / mL) to the negative control and positive control groups, so that the final C48 / 80 concentrations are 0 μg / mL, 10 μg / mL, and 20 μg / mL, respectively, and add each well at the same time. 1 μL Avidin-FITC (0.5 mg / mL). In addition, set up 1 tube of negative control group and 1 tube of positive control group separately...
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