Voie d'immunisation et séquence d'administration de l ... - TEL

tel-00827710, version 1 - 29 May 2013
2. Materials
2.1. Common reagents
1. Fresh or frozen sample (for mice, see Section 3.1; for humans, prepare PBMCs
according to standard procedures (see Note 1))
2. For humans studies only: fluorescently labeled mAb specific for MHC class I
molecules of interest, suitable for flow cytometry (for example anti-human HLA A2
antibody, BD Biosciences), and corresponding isotype (see Note 2)
3. 60mm-Petri dish
4. Falcon 15mL tube
5. 5mL FACS tubes
6. FcR blocking reagents
7. Anti-PE microbeads (see Note 3)
8. MACS separation columns, magnets, stands (see Note 4)
9. BD Falcon Cell Strainer 70µm
10. More than 5-colors flow cytometer, ideally with possibility to cell sorting
2.2 Buffers
1. PBS
2. PBS-2%FCS
3. Human Pulldown Buffer (HPB; ∼50 mL for one enrichment sample): PBS 1X, 5% of
Human Serum Albumin 20% (final concentration 1%), 5% Citrate Dextrose
Anticoagulant (see Note 5)
4. Mice Pulldown Buffer (MPB): PBS 1X, 2% FCS, 0.001% Sodium Azide
5. Mice Pulldown Buffer (MPB) without azide
6. Mice R-10 buffer: RMPI, 10% Fetal Calf Serum, 10mM HEPES, 1x Non Essential
Amino-Acids, 1mM Sodium Pyruvate, 60nM 2-Mercaptoethanol, 20 ng/mL
Gentamycin
2.3. Flow cytometry
1. PE- and/or APC- labeled MHC class I multimers (see Note 6 and 7)
2. For multiplexing experiments (determination of multiple specificities in one single
tube), biotinylated monomers (see Note 8) and streptavidin coupled to fluorochrome or
reporter of interest (PE-, APC-, PE-Cy7-, APC-Cy7-, Qdots-streptavidin, 1mg/mL)
3. Cocktail of fluorescently labeled mAb that are known to be expressed on cells you
wish to exclude from analysis (e.g., monocytes, B cells, and NK cells) (see Note 9).
These mAb should be coupled to a common fluorochrome, for example Pacific Blue,
Page 213 of 256