By Garry M. Walsh
In Eosinophil: equipment and Protocols, specialists within the box of eosinophil biology comprehensively offer exact methodological perception into the learn of this attention-grabbing phone. This booklet is geared toward a various diversity of simple and medical scientists who desire to paintings with eosinophils or who require an replace in their wisdom or to achieve the data required to check a functionality of the eosinophil assorted to their present quarter of enquiry. Written within the hugely profitable Methods in Molecular Biology series structure, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and key pointers on troubleshooting and averting recognized pitfalls.
Authoritative and sensible, Eosinophil: tools and Protocols seeks to assist scientist within the discovery of recent hypotheses and for additional exam of this fascinating cell.
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Extra info for Eosinophils: Methods and Protocols
Here, we describe a newly developed protocol for the purification of eosinophils from human bone marrow. Key words Bone marrow eosinophils, Purification, Human bone marrow, Cell isolation 1 Introduction Eosinophils are innate immune cells that play a role in anti-helminthic host defense and in allergic diseases such as asthma. The in vitro study of human eosinophils along with the characterization of their biological functions is most commonly achieved via their purification from peripheral blood using anti-CD16-conjugated-magnetic beads as described in Chapter 2.
Stir until dissolved. 3. 2 with 1 N NaOH or 1 N HCl. 4. Adjust the final volume (1 L) with water. 5. 22-μm filter using a positive pressure system. Aseptically dispense the medium into a sterile container. Finally, store the medium at 4 °C. 3 Chemical Inducer for the Differentiation of Eosinophils 3 1. Sodium butyrate: Dissolve in water. 5 M. Store at −20 °C (see Note 2). 1 Maintenance of HL-60 Clone 15 Cells and EoL-1 Cells HL-60 clone 15 cells may be obtained from the American Type Culture Collection, MD, USA (CRL-1964).
The final density of HL-60 clone 15 cells and EoL-1 cells is 1 × 105 cells/ml and 5 × 104 cells/ml, respectively. The final concentration of sodium butyrate is 500 μM. 9. Incubate at 37 °C under 5 % CO2 for 6 days. 10. After incubation, confirm that eosinophils have differentiated from the cell lines by staining using May-Grünwald Giemsa or Luxol fast blue and the levels of expression of cell surface molecules using a flow cytometer [5, 6] (see Note 3). 4 Notes 1. Eosinophilic differentiation of these cell lines is induced accompanied with growth arrest.
Eosinophils: Methods and Protocols by Garry M. Walsh