UK NEQAS LI was recently part of an international harmonized study for data analysis in the evaluation ofmultiple myeloma residual disease by high sensitivity flow cytometry. Read about the findings here:
For flow cytometry, cells were labeled with the following reagents: anti-SLAMF6 (NT-7), anti-CD45RA (HI100), anti-CCR7 (G043H7), anti-CD62 L (DREG-56), anti-CD8 (RPA-T8), anti-TNFα (Mab11), anti-CD244 (C1.7), anti-CD48 (BJ40), anti-CD229 (HLy-9.1.25), anti-CD84 (CD84.1.21), anti-CD4 (RPA-T4) were all obtained from BioLegend. Anti-SLAMF6 (REA) was from Miltenyi Biotec, anti-IFNγ (4S.B3) from Biogems, anti-FLAG (F3165) from Sigma-Aldrich, and anti-CD150 [A12(7D4)] from eBioscience.
The Jurkat cell line was purchased from ATCC in 2014. The cells were cultured in RPMI1640 supplemented with 10% heat-inactivated FCS, 2 mmol/L l-glutamine, and combined antibiotics. The line was regularly tested and was Mycoplasma free. The cells are authenticated once a year using flow cytometry markers. Cells grew in culture for a week (2 passages) before use.
The T2 cell line was a kind gift from the Surgery Branch in 2004. The cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mmol/L L-glutamine, and combined antibiotics. The line was regularly tested and was mycoplasma free. The cells are authenticated once a year using flow cytometry markers. Cells grew in culture for a week (2 passages) before use.
To confirm the existence of these isoforms, we analyzed a public RNA-sequencing bioproject (accession no. PRJNA482654; ref. 21). Using the RSEM package for quantifying isoform abundance, we identified all SLAMF6 RNA isoforms (Fig. 1D). We failed to identify exon2 splicing in the murine analogue of SLAMF6, Ly108, nor did we find data on murine isoforms in Ly108 extracellular domain from genome browsers (Supplementary Fig. S2A). In activated CD8+ T cells, SLAMF6 isoform 1, always expressed at higher levels compared with the shorter isoforms (ratio >1), was even further upregulated in effector and effector memory (EM) cells compared with naïve or central memory (CM) cells (Fig. 1E; Supplementary Fig. S2B and S2C). Flow cytometry confirmed that the expression of full-length SLAMF6 protein increased in parallel (Fig. 1F and G).
Considering colon cancer cell lines including SW480, SW620 and LoVo express different levels of biomarkers (e.g., EpCAM), which was confirmed by the flow cytometry (Additional file 1: Figure S1), the binding and capture capability of antiEpCAM and antiSlex sequentially-conjugated dendrimer conjugate (PE-5A-G6-5S-FITC) to the above EpCAM and Slex-expressing colon cancer cell lines was individually investigated by us.
To further explore how the G6-5A-5S conjugate affected the cell activity, cell cycle distribution was analyzed by flow cytometry. Cell lines after individual incubation with the conjugate for 48 h were stained with propidium iodide (PI) staining to determine the cell population in every phases of G0/G1, S and G2/M. Flow cytometric images showed that the conjugate could cause a concentration-dependent increase in cell population of the G0/G1 phase and a decrease in cell population of the S phase without a significant increase in cell population of the G2/M phase for SW620 cells (Figure 5a). Similar cell cycle distribution was found for SW480 cells. The significance between SW480 and SW620 cell lines indicated the increased inhibitory effect of conjugate on SW620 cells. However, for LoVo cells, the cell population in G2/M phase was concentration-dependently increased and that in S phase was decreased without a significant change in G0/G1 phase (Figure 5b), suggesting that dual antibody conjugate G6-5A-5S mainly arrested SW480 and SW620 cell lines at the G0/G1 stage and LoVo cells at the G2/M stage. The difference in cell cycle distribution might be attributed to the different interaction mechanism between dual antibody conjugate and each colon cancer cell line.
A Becton Dickinson (BD) multiparametric fluorescence-activated cell sorting (FACS) Aria III with laser excitation set at 488 was used for flow cytometric analysis. According to the forward versus side scatter histograms, gating strategy was used to set P1 gate for determining the target colon cancer cell lines. Fluorescence signals derived from PI (or PE) and DiOC6(3) (or FITC) were respectively detected through 585 and 530 nm bandpass filters. Side angle scattered light (SSC) versus PI histogram displayed the cell cycle distribution, SSC versus PE (or FITC) histogram showed the expression levels of biomarkers, SSC versus DiOC6(3) histogram revealed the cellular MMP, and PE versus FITC dot plots showed the captured cell numbers by the synthesized conjugate. All the data were acquired based on the collected 10,000 cells satisfying the light scatter criteria and analyzed using the BD FACS Diva software provided with the system.
Expression of hEag1 in leukemia cell lines and primary AML cells. A: hEag1 mRNA levels determined by real-time PCR, expressed as relative to that in UT-7 cells. B: hEag1 protein content in cell lines and primary cells from hEag1 positive patients measured by flow cytometry. The background rightward shift induced by anti-hEag1 antibody mAb49 in the PCR-negative P2 cells was set as zero value. C: hEag1 siRNA treatment (lower panels) strongly diminished the fluorescence shift attributable to hEag1 expression in PLB-985 and K562 cells. The gray peak indicates control secondary fluorescent antibody staining.
Protein expression was confirmed by flow cytometry using an anti-hEag1 specific monoclonal antibody (mAb49) directed against an extracellular loop of the channel. The results are summarized in Fig. 2B. Some background fluorescence was detected in all cell lines, including hEag1-negative patient samples. For this reason, we subtracted the magnitude of the mean fluorescence intensity (MFI) shift observed in Patient 2 cells (hEag1 negative in PCR) from all measurements. Under these conditions, hEag1 knockdown by siRNA in PLB-985 and K562 cells completely abolished the increase in fluorescence intensity (Fig. 2C), indicating that the MFI shift is due to hEag1 protein expression.
Finally, knockdown of hEag1 expression by siRNA in PLB-985 and K562 cells also resulted in up to 80% diminished proliferation over 5 days. (hEag1 knockdown by siRNA was not possible in UT-7 cells.) Representative proliferation curves are shown in Fig. 5. Effective hEag1 knockdown by siRNA was confirmed by flow cytometric detection of cells stained with anti-hEag1 mAb49 (see Fig. 2C). Altogether, our data suggest that hEag1 is implicated in the regulation of leukemia cell proliferation.
Cell cycle distribution and apoptosis in the presence of hEag1 inhibitors in vitro. A. The indicated cell lines were incubated with 4 μM astemizole (AST), 20 μM imipramine (IMI) or 10 μg/mL anti-hEag1 mAb56 for 2 days. Cell cycle phases were measured after staining with propidium iodide by flow cytometry. Only mAb56 increased significantly the proportion of cells in the S phase in K562 and HEL cells. B. K-562 and primary P4 cells were incubated with 4 μM astemizole or mAb56 antibody for 2 days, respectively and apoptosis was measured by flow cytometry. A clear increase in Annexin V fluorescence (apoptosis) was observed in both determinations.
As transient hEag1 expression is important during differentiation of other cell types like myoblasts, we tested its possible involvement in hematopoietic differentiation. We used the model cell line HL-60, which is arrested at the promyelocytic stage, but can be terminally differentiated over 6 days into granulocytes by retinoic acid. Cell differentiation was shown by Giemsa stain of cell nuclei and an increase of CD38 expression in flow cytometry measurements. We determined hEag1 expression every 2 h during differentiation but did not detect any hEag1 expression at any time during the entire differentiation process (data not shown).
Differentiation of HL-60 cells into granulocytes was induced with 2 μM retinoic acid over 6 days [35, 36]. During differentiation, samples were obtained every 2 h and analyzed for hEag1 expression up to day 6. Differentiation was monitored by successive Giemsa stains, and confirmed after 6 days by flow cytometry, through up-regulation of CD38 .
hEag1 silencing was performed using 30 nM siRNA (sense: r(CAG CCA UCU UGG UCC CUU A)dTdT, antisense: r(UAA GGG ACC AAG AUG GCU G)dTdA). Transfection was performed by nucleofection (Amaxa, Lonza, Cologne, Germany) according to the manufactures recommendations with 2 × 106 K562 or PLB-985. No transfection was achieved in UT-7 cells. Commercial non-targeting scrambled siRNA (Ambion, Darmstadt, Germany) and anti-GAPDH siRNA were used as negative and positive control, respectively. RNA and protein knockdown after 2 days was confirmed by real-time PCR and flow cytometry.
FcγRIIb on monocytes and B cells was determined by using anti-CD19-APC-Cy7, anti-CD14-Pacific Blue (BD Biosciences), anti-FcγRIIb-APC, and human IgG1 isotype control (BioInvent International AB). FcγRIIb expression was determined by using a FACSCanto II or FACSCalibur flow cytometer and was analyzed by using FCS Express (De Novo Software) or Cellquest (BD Biosciences).
FcγRIIb expression on monocytes is upregulated during HD preculture. (A) Histograms showing expression of FcγRIIb on monocytes from fresh PBMCs, and taken after 18 and 36 hours of HD preculture, and 36 hours of LD preculture, and on B cells from fresh PBMCs and after HD preculture. (B) Time course of the increase in FcγRIIb expression on monocytes during HD preculture. In contrast, B cells show no change in FcγRIIb expression during HD preculture. Bars show mean and range of 2 donors. Analysis in A and B was using a FACSCantoII. (C) Western blot showing an increase in the expression of the b2 isoform of FcγRIIb during HD preculture. (D) Comparison of FcγRIIb expression on fresh and HD precultured monocytes. (E) Plots showing relationship between FcγRIIb expression on monocytes after HD preculture and proliferation and TNFα release in response to TGN1412; n = 14, analyzed using 2-tailed Spearman rank correlation (Graphpad Prism 6). FcγRIIb expression and proliferation plots from all donors are shown in supplemental Figure 4. Analysis in D and E was using a FACSCalibur flow cytometer. 2b1af7f3a8