64 ANTI-AGEING
■ Cell viability(%)=OD 570sample group 570negative control group
×100%
■ Increase rate (%)=cell viability sample group - cell viability negative control group After culturing, the cells were gently washed
once with PBS and washed three times with PBS after fixing the tissue with fix solution for 15 minutes. Then, 100 mL of 0.1% Triton X-100 was added to each well for ventilation for 15 minutes, followed by three washes with PBS. After 45 minutes of blocking with 5% BSA,
100 μL of COL I antibody (diluted 1:100) was added to each well and incubated overnight at 4°C. The next day, the wells were washed three times with PBST after 60 minutes of rewarming at room temperature. Subsequently, 200 μL of anti-rabbit IgG
was added to each well and incubated in dark at room temperature for 60 minutes. Finally, photos were taken under the inverted fluorescence microscope after washing with PBST, and the photographic parameters were kept consistent.
■ Change rate (%) = (total fluorescence intensity sample group
group - total fluorescence intensity
control group) / total fluorescence intensity control ×100%
Nucleus damage Nucleus senescence changes were detected by immunofluorescence staining to carry out specific staining on key transcription factor HES1, DNA damage. After culturing, the culture medium was discarded and the samples were gently washed once with PBS. Following fixation of the tissue fix solution
for 15 minutes, the samples were gently washed with PBS three times. 100 microliters of 0.1% Triton X-100 was added to each well for ventilation for 15 minutes, followed by three washes with PBS. After a 45-minute blocking with 5% BSA, 100
μL of 8-OHdG antibody (1:30 dilution), γH2AX antibody (1:100 dilution), and HES1 antibody (1:100 dilution) were added to each well respectively, and the samples were incubated overnight at 4°C. The next day, the wells were washed three
times with PBST after 60 minutes of rewarming at room temperature. Subsequently, 200 μL of anti-mouse IgG was added to each well and
P7 UVAP7
1.2 1.0 0.8 0.6 0.4 0.2 0.0
/OD
48h +300.39%
*** *** ** *** ***
***
UVA-P7 + BF Compared with UVAP7, * means p<0.05, ** means P<0.01, *** means p<0.001 Figure 2: The OD Value of cell viability
incubated in dark at room temperature for 60 minutes. Finally, photos were taken under the inverted fluorescence microscope after washing with PBST, and the photographic parameters were kept consistent.
■ Change rate (%) = (total fluorescence intensity sample group
total fluorescence intensity control group
- total fluorescence intensity control group ×100%
) /
Mitochondrial activity The changes of mitochondrial activity were detected by staining with the MitoBright LT probe. After culturing, the culture medium was discarded, and 100μL of MitoBright LT working solution was added to each well for staining for 30 minutes. Following two washes with PBS,
photographs were taken under the inverted fluorescence microscope. Excitation was set at 640nm, and emission ranged from 650- 700nm, ensuring consistency in photographic parameters.
■ Change rate (%) = (total fluorescence intensity sample group
group - total fluorescence intensity
control group) / total fluorescence intensity control ×100%
UVAP7+0.156% BF UVAP7+0.3125% BF UVAP7+0.625% BF
20000 15000 10000 5000 0
Senescence-associated secretory phenotype The changes of MMP-3 were detected by ELISA method. After culturing, the cell culture supernatant was collected and the experimental operation was carried out according to the standard operation procedure of ELISA kit. The OD value was detected at 450 nm on
the multi-function microplate reader, and the content of MMP-3 and its inhibition rate were calculated according to the standard curve.
■ Inhibition rate (%) = (content control group content sample group
)/content control group - × 100%
Clinical tests Test product and participants The test product was prepared in the laboratory. Bifida Ferment Lysate at the concentration of 3% was added to the base serum to form a transparent serum. All ingredients have passed the safety assessment. Thirty-two healthy participants were
recruited, two of whom withdrew from the test for personal reasons. The age range of the remaining 30 participants was 37 to 58 years old, with an average age of 47.9 years old.
+843.93% *** -66.23% *** *** *** ***
* Red florescence-COL I
Cell: Natural aging cells (P7), photoaging cells (UVAP7) Figure 3: Fluorescent photos of COL I PERSONAL CARE November 2024 Figure 4: Fluorescence intensity of COL I
www.personalcaremagazine.com
UVA-P7 + BF
OD570
COL I RFU
P7 UVA-P7 0.01% 0.03% 0.1% 0.3% 1% 3%
P7 UVA-P7 0.078% 0.156% 0.3125% 0.625% 1.25%
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