Hey Dr. Dear

Look attached , I hope helping you.

Good luck

Dear Awatif

Thank you very much for your help. Really much appreciated.

Shilan

Stain the plasma membrane with fluorescently-labeled cholera toxin dye (e.g labeled  with AlexaFlour -647 which emits in the Far Red channel   and count the number of labelled zymosan particles (labeled in a different channel colour other than the one you l used for labeling the cell membrane , in your case Alexa-546). Then permeabilize the cell membrane by a detergent-based solution e.g 0.1 %  triton TX-100, then label the phagocytosed/internalized zymosan particles with a 3rd channel colour e.g  FITC (green) . Then calculate the phagocytic index by counting  (for a 100 cells) the number of zymosan particles internalized versus total Zymosan (in +out)

Phagocytic index %:  Zym  (in)/Total Zymosan particles x100

Hope that helps !

Dear Amira

Thank you very much for your help. yes, Iam doing zymosan(red) which is AlexaFluoro 594. with phalloidin (FITC) green which stains the actin. thank you for the phagocytic index as well much appreciated

You are most welcome...  Yeah I have done  actin phalloidin staining many times...Yup I mixed up the colours , but you still need to stain the membrane with  a cholera toxin labeled with Alexa 647 (cy5 channel)

Good Luck!

Ok Iam going to order the membrane stain that you recommended.

Many thanks

Please check artcles by Richard stokes and David Speert

such as

http://www.jimmunol.org/content/160/11/5514.full.pdf

Dear Sameer

Thank you very much for your help. much appreciated.

Your welcome, hope it was helpful