easysep human whole blood cd34-positive selection kit  (STEMCELL Technologies Inc)

Journal: PLoS ONE

Article Title: SALL4, a Stem Cell Factor, Affects the Side Population by Regulation of the ATP-Binding Cassette Drug Transport Genes

doi: 10.1371/journal.pone.0018372

Figure Lengend Snippet: ( A ) 293 cells were transiently transfected with SALL4A or SALL4B or pcDNA as a control. At 48 hr after transfection, RNA was isolated from transfected cells. Transfected cells were subjected to qRT-PCR to measure the mRNA expressions of ABCA3 and ABCG2. The mRNA expressions of ABCA3 and ABCG2 were significantly increased by SALL4. Data are represented as mean ± S.D. from three independent experiments. *** denotes p<0.001, ** p<0.01, *p<0.05. ( B ) CD34+ cells were transduced with retrovirus expressing shRNA against SALL4 and subjected to qRT-PCR analysis to measure the expression levels of ABCA3 and ABCG2. The expression level of ABCA3 was reduced by 80% and the expression level of ABCG2 was reduced by 74% in CD34+ cells compared to those of the controls. The mRNA level was normalized with the internal control GAPDH. ( C ) qRT-PCR analysis of SALL4A and B, ABCG2, and ABCA3 in HL-60, KG1a, and RPMI8226 cells. For quantification of gene expression, amplification of GAPDH was performed as an endogenous control to standardize the amount of sample. ( D and E ) SALL4 and ABC gene expressions were correlated in primary AML samples. 461 primary AML samples (GSE6891) were stratified into SALL4 high and SALL4 low using the mean intensity of SALL4 as a threshold. ( F and G ) qRT-PCR was performed on 34 primary samples with primers for SALL4, ABCG2 and ABCA3. Mean value of SALL4 was used to stratify the samples to SALL4 high or SALL4 low groups. Data are represented as mean ± S.D. from three independent experiments. Y axis: Relative SALL4 expression when compared to controls.

Article Snippet: The cells were then sorted using the EasySep Human Whole Blood/Buffy Coat CD34 Positive Selection Kit (Stemcell Technologies, Vancouver, Canada).

Techniques: Transfection, Isolation, Quantitative RT-PCR, Transduction, Expressing, shRNA, Amplification

Journal: Oncotarget

Article Title: Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

doi: 10.18632/oncotarget.14324

Figure Lengend Snippet: A . 17-AAG and CP suppressed the self-renewal of primitive progenitors (LTC-ICs). After treating with 17-AAG and CP at the indicated concentrations for 24 h, MNCs from CML bone marrow were examined by LTC-ICs assay. The percent inhibition of LTC-ICs proliferation in 17-AAG and CP treated group relative to untreated controls was shown (CML, n = 3). B . Representative data for CML primitive and CML committed progenitor apoptosis. After treating with 17-AAG and CP at the indicated concentrations for 24 h, CML CD34 + CD38 - primitive and CD34 + CD38 + committed progenitors were incubated with Annexin V-FITC solution. The Annexin V positive cells were evaluated by FACS. C . The scheme of the origins of the LSCs and the effects of 17-AAG+CP on LSCs via eradicated leukemia cells at different mature states.

Article Snippet: CD34+ cells were isolated by EasySep® Human Whole Blood CD34 Positive Selection Kit ((#18096, StemCell Technologies, Canada) and seeded in MethoCult® GF H4434 medium (StemCell Technologies, Canada) as previously described [ ], the CFU cells were scored after 10-14 days.

Techniques: Inhibition, Incubation

Journal: Oncotarget

Article Title: Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

doi: 10.18632/oncotarget.14324

Figure Lengend Snippet: A . Treatment with 17-AAG, CP, 17-AAG+CP for 24 h, CP 3 h→17-AAG 24 h, 17-AAG 3 h→CP 24 h in cells retarded signal pathway initiated by Bcr-Abl. Thirty micrograms of cytosolic protein were resolved, transferred to a PVDF membrane, and then probed the level of p-c-Abl, p-Stat5, and p-CrkL. eIF4E served as input control. B . Band densities were measured and normalized to eIF4E. *p<0.05, ** p<0.01 vs control. C . Treatment with 17-AAG, CP, or 17-AAG+CP for 24 h has potent inhibition of Bcr-Abl initiating signaling in CD34+ cells from the bone marrow of CML patients. D . Band densities were normalized to eIF4E. *p<0.05, ** p<0.01 vs control. Note: 17-AAG: 1 μM 17-AAG; CP: 5 μM CP.

Article Snippet: CD34+ cells were isolated by EasySep® Human Whole Blood CD34 Positive Selection Kit ((#18096, StemCell Technologies, Canada) and seeded in MethoCult® GF H4434 medium (StemCell Technologies, Canada) as previously described [ ], the CFU cells were scored after 10-14 days.

Techniques: Membrane, Control, Inhibition

Journal: Oncotarget

Article Title: Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

doi: 10.18632/oncotarget.14324

Figure Lengend Snippet: A . CFU assay on progenitor cells from CML bone marrow. Enriched CML (n =3) CD34+ cells were treated with or without graded concentrations of 17-AAG and CP for 24 hours and further cultured for 14 days, then CFU cells were scored. B . Trypan blue exclusion measured the viable and dead CML CD34+ cells. After treating for 24 h, CD34+ cells were mixed with trypan blue and then the cell viability were determined. C . CFU assay ofimatinib-resistant CML CD34+ cells. ** p<0.01 vs IM 0.8 μM group. D . CFU assay on normal CD34+ cells. Values were expressed as the percentage compared to controls.

Article Snippet: CD34+ cells were isolated by EasySep® Human Whole Blood CD34 Positive Selection Kit ((#18096, StemCell Technologies, Canada) and seeded in MethoCult® GF H4434 medium (StemCell Technologies, Canada) as previously described [ ], the CFU cells were scored after 10-14 days.

Techniques: Colony-forming Unit Assay, Cell Culture