Dear Dr. Gupta,I have a question about your protocol. If I want...

Dear Dr. Gupta,

Thanks a lot for your reply. I'm sorry to bore you again. I have 2 questiones.

1. Why you remove the water bottle for 5-6 h or overnight before the FITC-dextran oral gavage? In some papers there are no mention of water starvation.

2. What kind of 96-well microplate are you used? Black or white? Do we need transparent bottom of the microplate?

Best wishes.

Dear LI,
1. We kept samples at 4 ºC for maximum of 2h after taking it out from mice and immediately analyzed for FITC determination by spectrophotometer.
2. As I said we always used fresh serum to analyze FITC concentration in serums but in theory if you protect from light and samples are nicely and similarly stored at -20ºC, you should be able to detect FITC with minimum loss of its Fluorescence.
3. I can’t exactly answer your question about how long it will take to clear out FITC-dextran from blood. This you should check. You do not need to kill mice after 4h if you are able to take around 250ul blood to get atleast 100 ul serum.
best,
Jalaj

Dear Dr. Gupta,
Thanks for your reply. I have some more questions.
1. How long can I keep the serum samples at 4 °C until the spectrophoto determination?
2.Can I store the serum samples at -20°C or -80°C until I collect enough samples for spectrophoto determination? Will it change the activity of fluorescein isothiocyanate?
3. How long can the FITC-dextran clear out of body compeletely?After oral gavage of FITC-dextran for 4h, can I collect serum sample but not kill the mice, keep the mice for somedays and gavage FITC-dextran again and collect serum sample?
Thank you a lot for your reply.

Best wishes!

Dear LI,
Thank you for your interest in our protocol. You can use only one standard curve and serum from one of the untreated WT mice of same genetic background (better littermate) to determine the concentration of FITC-dextran in the serum samples from both WT and IL6 KO mice.
I hope this will help you.
Good Luck,
Best,
Jalaj Gupta