Prepare media for culture 3T3-L1 from deep freezer stock and
Lactobacillus species
June 24th
Check whether 3T3-L1 grows well and media change
June 26th
Split the 3T3-L1
June 28th
Seed the 3T3-L1 into 12 wells and prepare the media to grow
Lactobacillus species
July 2025
July 1st
Confirm growth arrest of 3T3-L1 and put the Lactobacillus species at
appropriate MOI
July 2nd
Put the Gentamycin at appropriate concentration
July 4th
Change the induction media 1 with Lactobacillus spp.
July 5th
Put the Gentamycin at appropriate concentration
July 8th
Change the induction media 2 with Lactobacillus spp.
July 9th
Put the Gentamycin at appropriate concentration
July 11th
Oil red O staining, capture the phenotype image of each species and
measure the 518 wavelength
July 12th
Prepare the supernatant of Lactobacillus spp.
July 13th
Isolation the Exosome from each spp.
July 14th
Put the supernatant and exosome into the 3T3-L1 adipocyte during
adipogenesis
July 17th
Change the induction media 1 with supernatant and exosome
July 19th
Change the induction media 2 with supernatant and exosome
July 21st
Change the FBS media with supernatant and exosome
July 23rd
Oil red O staining, capture the phenotype images and measure the 518
wavelength
Isolate the RNA and protein from the cells
Do the proteomics analysis of samples from exosome filtering and
non-exosome filtering
July 24th
Order the hisF sequence of Lactobacillus rhamnosus to synthesis provider
Convert cDNA from samples and running the qPCR analysis
Gel electrophoresis of proteins from the samples and blocking with 5%
skim milk and put the primary antibody of adipogenesis markers with
housekeeping genes
July 25th
Put the secondary Ab and take the WB images
Digest restriction enzyme with pET28b9(+) vector and insert sequence
Gel electrophoresis whether vector and genes are cut well
July 26th
Insert the genes into the plasmid and heatshock to competent cells top
10 and BL21(DE3)
July 27th
Check the colonies with selection media
July 28th
Check the sequence of the colonies whether plasmid with sequence is well
cloned
July 29th
Express the protein at appropriate IPTG concentration and temperatures
within cell system
Express the protein in cell-free system
August 2025
August 2nd
Lyse the expressed cells by sonication with protein media and change the
media with PBS
Collect the protein of cell-free system and change the media with PBS
Seed the 3T3-L1 adipocyte into 12 wells
August 5th
Put the purified hisF at appropriate concentration into adipocyte during
adipogenesis
August 8th
Change induction media 1 with hisF
August 10th
Change the induction media 2 with hisF
August 13th
Change the induction media 3 with hisF
August 15th
Oil red O staining, capture the phenotype images and measure the 518nm
wavelength
Collect the RNA and protein samples from matured adipocytes
