Eosinophils: Methods and Protocols by Garry M. Walsh
By Garry M. Walsh
In Eosinophil: tools and Protocols, specialists within the box of eosinophil biology comprehensively offer unique methodological perception into the examine of this attention-grabbing mobilephone. This e-book is geared toward a various variety of uncomplicated and medical scientists who desire to paintings with eosinophils or who require an replace in their wisdom or to achieve the data required to review a functionality of the eosinophil various to their present sector of enquiry. Written within the hugely profitable Methods in Molecular Biology series structure, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols, and key pointers on troubleshooting and heading off recognized pitfalls.
Authoritative and functional, Eosinophil: equipment and Protocols seeks to help scientist within the discovery of latest hypotheses and for additional exam of this interesting cell.
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2. Perform the appropriate compensations. Compensation of the surface markers can be performed with either single stained populations or with the appropriate beads (see Note 13). 3. Compensation for 7-AAD as a DNA marker needs to be performed with cells fixed with BD Cytofix/Cytoperm, so the dye can pass through the cell membrane and incorporated into the DNA. 1 ml BD Cytofix/ Cytoperm. Incubate for 15 min at RT. Wash cells as above (1 ml of 1× BD Perm/Wash Buffer, centrifuge at 200 × g for 10 min at 4 °C, decant).
Compensation of the surface markers can be performed with either single stained populations or with the appropriate beads (see Note 13). 3. Compensation for 7-AAD as a DNA marker needs to be performed with cells fixed with BD Cytofix/Cytoperm, so the dye can pass through the cell membrane and incorporated into the DNA. 1 ml BD Cytofix/ Cytoperm. Incubate for 15 min at RT. Wash cells as above (1 ml of 1× BD Perm/Wash Buffer, centrifuge at 200 × g for 10 min at 4 °C, decant). Add 7-AAD dye to the cells that will serve as a positive control for the compensations.
2× PBS: Dilute 10× PBS fivefold to make 2× PBS. 4. Cold sterile water. 5. RPMIeos: RPMI 1640 supplemented with 100 U/mL penicillin G, 10 μg/mL streptomycin, 3 μg/mL ι-glutamine, 50 μM β-mercaptoethanol, 20 % heat-inactivated fetal calf serum (FCS), and 1 ng/mL human recombinant IL-5. 6. 75 cm2 tissue culture flask. 7. 10 cm tissue culture dish. 8. 15- and 50-mL conical tubes. 3 Methods A summary of the purification protocol is depicted as a flowchart in Fig. 1. Perform all steps in a sterile tissue culture room or a designated sterile ventilated tissue culture hood.