By Stephen P. Robinson, Andrew J. Stagg
Stephen P Robinson, MD, PhD, and Andrew Stagg, PhD, have introduced jointly quite a lot of time-proven tools for learning those dendritic cells. lots of those without difficulty reproducible options take care of the matter of acquiring adequate dendritic cells for research, no matter if via isolation from a large vareity of tissues, or from quite a few progenitor telephone populations. different equipment describe in step by step type the concepts common for reading points of dendritic cells, starting from cellphone migration to antigen uptake and T mobile stimulation. additionally, a number of concepts discover the sensible demanding situations taken with utilizing dendritic cells in a scientific environment to increase novel immunotherapeueutics.
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Extra resources for Dendritic Cell Protocols
8. 9. 10. 11. 12. 13. 14. Specific pathogen-free rats of any strain, at 10-12 weeks of age. Sterile Petri dishes. Curved forceps and straight forceps with serrated ends. Hanks balanced salt solution (HBSS) with Ca++, Mg++. HBSS without Ca++, Mg++. 5mg/ml DNAase (Boehringer Mannheim) in HBSS (with Ca++, Mg++). Keep on ice and filter sterilise just before use. 5mg/ml) can be stored frozen until required (see Note 5). Collection buffer: 20mM EDTA in HBSS (without Ca++, Mg ++). The EDTA inhibits the enzyme activity by chelation of Ca++.
As was pointed out in the description of experimental steps, keratinocytes sometimes stick to adherent Langerhans cells on the antibody-coated dishes. This phenomenon can be monitored directly under phase contrast and the extent of washing can be adjusted accordingly. This kind of monitoring is not possible with FACS and immunomagnetic methods. With regard to “antibody loading” of selected Langerhans cells panning, FACS and magnetic methods are alike: In each method Langerhans cells still carry the primary (antiMHC class II or other) mAb on their surface.
When no monolayer forms, one can anticipate low yields of cultured Langerhans cells. It takes a good phase contrast to see the monolayer. It can be better appreciated after having washed off the adherent cells. So, at the end of the culture period, keep the dishes and check them! 4. Upon close and patient inspection under a phase contrast microscope one can also spot occasional “veiled” Langerhans cells on d 2 and 3 of culture. They are either lying on top of spread keratinocytes or they are floating freely.