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Blood, Vol. 113, Issue 3, 668-670, January 15, 2009
In vivo expansion of cells expressing acquired CSF3R mutations in patients with severe congenital neutropenia
Blood Germeshausen et al.
113: 668
Supplemental materials for: Germeshausen et al
Monoclonal Antibodies
The following antibodies were used for characterization and sorting of hematopoietic subsets: FITC conjugated CD3 (UCHT1), CD13 (SJ1D1), CD33 (D3HL60.251, Immunotech), CD52 (YTH 34.5), Phycoerythrin conjugated CD15 (VIMC6), CD19 (SJ25-C1), PE-Cy5 conjugated CD16 (3G8), biotinylated CD14 (Tuek4, Caltag), APC conjugated CD34 (8G12, Becton Dickinson). Streptavidin-PE-Cy7 (BD Pharmingen) was used for detection of biotinylated CD14. Cell Sorting
Cell sorting was performed on FACSAria (Becton Dickinson) and MoFlo (DakoCytomation) cell sorters. The following subsets were sorted from bone marrow samples of the patients: leukemic blasts (Pt 2 only, SSClowCD34+CD38−), hematopoietic progenitors (SSClowCD34+CD14−CD15−, additionally for Pt 2: CD38+), myeloid progenitors (Pts 3 and 4 only, CD13/CD33+CD34−CD14−CD15−), granulocyte progenitors (promyelocytes-myelocytes, CD15+CD16−), monocytes (CD14+CD15−CD16−), neutrophils (Pts 2–4 only, CD15+CD16+CD14−), T cells (CD3+CD19−) and B cells (CD19+CD3−). Cells were sorted twice: in a first round we sorted batches of the populations defined above (FACSAria), in a second round single cells were sorted into 15 µL of proteinase K lysis buffer (MoFlo). PCR
After lysis, proteinase K digestion and inactivation of proteinase K, the single cell lysates underwent a primary and secondary PCR and direct sequencing. The amplified fragment spanned the mutation sensitive region 2342–2541 of the CSF3R gene (fw: 5′-AACAGCTCAGAGACCTGTGGCCT-3′, rv: 5′-CCAAGGGGCTGGCCTGGA-3′). The primers for the secondary PCR were elongated for 6 nucleotides 3′. We used a mixture of DNA polymerases Taq and Pfu for PCR amplifications using 10 cycles with 68°C annealing temperature followed by 40 cycles with 64°C for primary and secondary PCR. DMSO was added with 1.2% (vol/vol) for the primary, and 2% for the secondary PCR.
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