Supporting Information Polyplex Exposure Inhibits Cell .
S1Supporting InformationPolyplex Exposure Inhibits Cell Cycle, Increases Inflammatory Response, and Can CauseProtein Expression Without Cell DivisionRebecca L. Matz,1,2 Blake Erickson,2,3 Sriram Vaidyanathan,2,5 Jolanta F. Kukowska-Latallo,2,4James R. Baker, Jr.,2,4,5 Bradford G. Orr,2,6 Mark M. Banaszak Holl*,1,2,3,51Department of Chemistry, 2Michigan Nanotechnology Institute for Medicine and BiologicalSciences, 3Program in Biophysics, 4Department of Internal Medicine, 5Department ofBiomedical Engineering, 6Department of Physics, University of Michigan, Ann Arbor, MI 48109ExperimentalPKH26 Staining – Rationale for Short Incubation Time Prior to TransfectionIn order to maximize the dynamic range of the PKH26 fluorescence signal in theseexperiments, the amount of incubation time prior to transfection was minimized. The cell linesused in these experiments adhered very well to the tissue culture dishes, and throughout theexperiments there were no qualitative differences in the adherence of these cell lines ascompared to when the seeding times were longer. The control samples (PKH26 only) in Figures1, 3, S2, and S5 yield a regular doubling time, demonstrating that these seeding times weresufficient to give normal cell division behavior. The data shown in Figure S1, obtained by amethod independent of that used in the aforementioned figures, also shows a normal celldoubling time.Cell Proliferation Assay (Figure S1)To test whether the proliferation rate of HeLa S3 cells changed upon staining withPKH26, cells were either stained with PKH26 or taken through the staining procedure albeit withno PKH26 present, plated at 80,000 cells/well in 12-well plates with 800 µL complete medium,and incubated for 5 to 6 h at 37 C with 5% CO2 as described. Cells were harvested at varioustime points over 2 days for flow cytometry also as previously described with the exception that7AAD was not employed as a viability stain. The density of the cell suspensions were countedwith a BD Accuri C6 Flow Cytometer (Accuri Cytometers; Ann Arbor, MI) which reports theamount of cell suspension volume pulled for a given number of cells, and curves of bestexponential fit were again determined using Microsoft Excel . The experiment was repeatedthree independent times.
S2pDNA Uptake Assay (Figure S3)To test the amount of pDNA uptake in HeLa S3 and 293A cells, cells were taken throughthe PKH26-staining procedure with no PKH26 present, plated at 80,000 cells/well in 12-wellplates with 800 µL complete medium, and incubated for 5 to 6 h at 37 C with 5% CO2 asdescribed. Cells were transfected for 3 h as described with polyplexes formed between jetPEITMand blank pDNA labeled with CX-rhodamine (Cat. No. MIR 3125; Mirus Bio; Madison, WI)according to the manufacturer’s protocol. After 9 h further incubation in complete medium, thecells were harvested as described and analyzed for CX-rhodamine fluorescence using a BDAccuri C6 Flow Cytometer . See Discussion S1 for full details on data interpretation.Cellular Senescence Assay (Figure S6)HeLa S3 and 293A cells were plated in triplicate and transfected as described except thatcells were not stained with PKH26 and the experiment was performed in 24-well tissue culturetreated plates (Cat. No. 353047; Becton, Dickinson and Company); all reagents and cell numberswere scaled down according to the growth area of the wells. Following exposure to jetPEITMonly or polyplexes formed between jetPEITM and the five types of DNA, cells were tested forcellular senescence (Cat. No. KAA002; EMD Millipore; Billerica, MA) per the manufacturer’sinstructions at 48 h. The positive control was cells that had been exposed to 50 nM doxorubicinhydrochloride (Cat. No. 2004; AvaChem Scientific; San Antonio, TX) from 3 days prior totransfection through the time point at which cells were assayed for senescence (i.e., for 5 daystotal). This procedure assesses the activity of senescence-associated β-galactosidase (SA-β-gal)that is identified by blue-green stained cells under light microscopy. Brightfield images wereobtained with an Olympus iX70-S1F2 microscope (Olympus Corporation; Tokyo) fitted with anOlympus DP72 digital camera, and the percent of blue-green as compared to normal cells acrosssix separate images for each sample was counted according to previously establishedprotocols.1,21. Dimri, G. P.; Lee, X.; Basile, G.; Acosta, M.; Scott, G.; Roskelley, C.; Medrano, E. E.;Linskens, M.; Rubelj, I.; Pereira-Smith, O. A biomarker that identifies senescent human cells inculture and in aging skin in vivo. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9363-9367.2. Schwarze, S. R.; Fu, V. X.; Desotelle, J. A.; Kenowski, M. L.; Jarrard, D. F. TheIdentification of Senescence-Specific Genes during Induction of Senescence in Prostate CancerCells. Neoplasia 2005, 7, 816-823.
S3Figure S1. Representative data showing that, as measured by density of cell suspensions,staining with PKH26 does not affect the proliferation rate of HeLa S3 cells as compared tocontrol cells. Each point shows the mean SD of three technical replicates with curve of bestexponential fit. The differences in absolute numbers of stained vs unstained cells is likely due tonormal variation in cell counting prior to plating. The inset shows summary of data from threeindependent experiments. M 26.0, SE 0.79 for PKH26-stained cells as compared to M 26.4,SE 0.92 for control cells, t(4) 0.30, p 0.05.Figure S2. PKH26 intensity of representative 293A cells treated with polyplexes formedbetween jetPEITM and CFP pDNA over 2 days following transfection as compared to controlcells (PKH26 only). Cells expressing CFP ( ) and those not expressing CFP ( ) are subsets ofthe whole population ( ) that was treated with polyplexes. Cells were transfected for 3 hbeginning at time 0 h. Each point shows the mean SD of three technical replicates with curveof best exponential fit. All data are fit with PKH26 only data at 0 h.
S4Figure S3. CX-rhodamine fluorescence intensity for representative HeLa S3 and 293A cellstreated with polyplexes formed between jetPEITM and CX-rhodamine-labeled blank pDNA. Cellswere transfected for 3 h and incubated 9 h further before being harvested and analyzed. ForHeLa S3 and 293A cells the entire population of cells exhibits a mean fluorescence shift of 4500 240 and 6200 390, respectively, consistent with a substantial degree of transfection for allcells in the population. The mean fluorescence shifts are the mean SD of three technicalreplicates. See Discussion S1 for full details on data interpretation.Figure S4. Representative schemes showing the approximate breakdown of divided vs notdivided and expressing vs not expressing cells 48 h following transfection. Division ismeasured by dilution of the membrane-stable dye PKH26, and expression is measured byfluorescence of the plasmid-encoded protein. The percentages are taken from the data shown inTable 1 (HeLa S3, Expt #1; 293A, Expt #3).
S5Figure S5. Representative data for (a) HeLa S3 and (b) COS-7 cells showing that exposure topolyplexes slows the doubling time. (c) Representative data for HeLa S3 cells showing thatexposure to the pDNA alone did not affect the doubling time. The cells were transfected for 3 hbeginning at time 0 h. Each point shows the mean SD of three technical replicates with curveof best exponential fit except for the cells only sample, which has a linear line of best fit. All dataare fit with PKH26 only data at 0 h except for the cells only sample. (d) Average PKH26intensity of untreated and CFP pDNA (CMV) polyplex-treated HeLa S3 cells over time based onthe average doubling times shown in Figure 3b. The standard deviations reflect the standarddeviations of the average doubling times.
S6Figure S6. HeLa S3 (a) and 293A (b) cells do not enter cellular senescence upon exposure to thepolymer alone (jetPEITM only) or any type of polyplex used in this study. Each data point islabeled with N, the number of cells counted for that sample. N for the doxorubicin case is lowerthan for the other treatments because there were fewer cells per image due to doxorubicininduced toxicity. According to Fisher’s exact test, no samples are more senescent than the cellsonly negative control, except for the 50 nM doxorubicin positive control. *** p 0.01.
S7
S8Figure S7. Scatterplots showing the relative number of up- ( ) and downregulated ( ) genes forthe jetPEITM, GFP pDNA (CMV) polyplex, and GFP pDNA (EF1α) polyplex treatmentscompared to the untreated control at 4 h following transfection. The GFP pDNA (EF1α) polyplextreatment at 24 h is also shown. (a) – (d) Cell cycle array. (e) – (h) Inflammatory response andautoimmunity array. The centerline indicates a fold change (2- Ct) of 1, and the upper and lowerlines indicate fold changes of 2 in gene expression. Circles indicate fold changes 2. Comparingto the untreated control, filled symbols indicate regulation that is significantly different (p 0.05), and open symbols indicate regulation that is not significantly different. Only thesignificantly up- or downregulated genes shown in Tables 2 and 3 are labeled in this figure. Notethat the cell cycle array data for the 24 h treatment with GFP pDNA (EF1α) polyplexes is basedon only two biological replicates; the third had to be removed due to evident inhibition of thereverse transcription step.
S9Table S1. Average doubling times of HeLa S3 and 293A cells exposed to jetPEITM only, orpolyplexes made between jetPEITM and ssDNA or various pDNAs.Average Doubling Time (h)aHeLa S3293ATreatmentPKH26 only (neg. control)25 225 3jetPEITM only29 224 4ssDNA polyplexes31 1 **52 9 **CFP pDNA (CMV) polyplexes53 8 ***46 9 **Blank pDNA (CMV) polyplexes 52 9 **35 4 ***GFP pDNA (CMV) polyplexes 49 861 10 *GFP pDNA (EF1α) polyplexes52 1045 16a* p 0.10, ** p 0.05, *** p 0.01.
S10Table S2. Changes in expression of all genes related to the cell cycle pathway in jetPEITM orpolyplex-exposed HeLa S3 cells.GeneBankNM 005157NM 013366NM 013367NM 000051NM 001184NM 004324NM 016567NM 000633NM 001168NM 007294NM 000059NM 031966NM 004701NM 005190NM 053056NM 001759NM 001238NM 001761NM 004060NM 004354NM 001239NM 001240NM 001241NM 003903NM 001786NM 001255NM 004359NM 001798NM 000075NM 003885NM 016408NM 001259NM 001799NM 001260NM 000389NM 004064NM 000077NM 004936NM 005192NM 001274NM 007194NM 001826NM 001827NM 003592NM 003591NM 003590NM 004399NM 004675NM 004945NM 001950NM 001924NM 005316DescriptionaC-abl oncogene 1, receptor tyrosine kinaseAnaphase promoting complex subunit 2Anaphase promoting complex subunit 4Ataxia telangiectasia mutatedAtaxia telangiectasia and Rad3 relatedBCL2-associated X proteinBRCA2 and CDKN1A interacting proteinB-cell CLL/lymphoma 2Baculoviral IAP repeat-containing 5Breast cancer 1, early onsetBreast cancer 2, early onsetCyclin B1Cyclin B2Cyclin CCyclin D1Cyclin D2Cyclin E1Cyclin FCyclin G1Cyclin G2Cyclin HCyclin T1Cyclin T2Cell division cycle 16 homolog (S. cerevisiae)Cell division cycle 2, G1 to S and G2 to MCell division cycle 20 homolog (S. cerevisiae)Cell division cycle 34 homolog (S. cerevisiae)Cyclin-dependent kinase 2Cyclin-dependent kinase 4Cyclin-dependent kinase 5, regulatory subunit 1 (p35)CDK5 regulatory subunit associated protein 1Cyclin-dependent kinase 6Cyclin-dependent kinase 7Cyclin-dependent kinase 8Cyclin-dependent kinase inhibitor 1A (p21, Cip1)Cyclin-dependent kinase inhibitor 1B (p27, Kip1)Cyclin-dependent kinase inhibitor 2A (melanoma, p16,CDKN2Ainhibits CDK4)Cyclin-dependent kinase inhibitor 2B (p15, inhibitsCDKN2BCDK4)CDKN3Cyclin-dependent kinase inhibitor 3CHEK1CHK1 checkpoint homolog (S. pombe)CHEK2CHK2 checkpoint homolog (S. pombe)CKS1BCDC28 protein kinase regulatory subunit 1BCKS2CDC28 protein kinase regulatory subunit 2CUL1Cullin 1CUL2Cullin 2CUL3Cullin 3DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 11DDX11(CHL1-like helicase homolog, S. cerevisiae)DIRAS3DIRAS family, GTP-binding RAS-like 3DNM2Dynamin 2E2F4E2F transcription factor 4, p107/p130-bindingGADD45A Growth arrest and DNA-damage-inducible, alphaGTF2H1General transcription factor IIH, polypeptide 1, CDK5RAP1CDK6CDK7CDK8CDKN1ACDKN1BFold Change in Gene ExpressionbGFP (CMV)GFP 61.03.5-1.0-1.71.51.05.51.3-1.23.8-1.0
S11NM 016426NM 016323NM 004507NM 014708NM 002266NM 002358NM 006341NM 004526NM 002388NM 005914NM 006739NM MCM4MCM5MKI67NM 002431MNAT1NM 005590MRE11ANM 002485NM 182649NM 002853NM 002873NM 002875NM 004584NM 000321NM 002894NM 002895NM 005611NM 002947NM 013376NM 005983NM 003352NM 007111NM 006286NM 000546NM PA3SERTAD1SKP2SUMO1TFDP1TFDP2TP53UBA1G-2 and S-phase expressed 1Hect domain and RLD 5HUS1 checkpoint homolog (S. pombe)Kinetochore associated 1Karyopherin alpha 2 (RAG cohort 1, importin alpha 1)MAD2 mitotic arrest deficient-like 1 (yeast)MAD2 mitotic arrest deficient-like 2 (yeast)Minichromosome maintenance complex component 2Minichromosome maintenance complex component 3Minichromosome maintenance complex component 4Minichromosome maintenance complex component 5Antigen identified by monoclonal antibody Ki-67Menage a trois homolog 1, cyclin H assembly factor(Xenopus laevis)MRE11 meiotic recombination 11 homolog A (S.cerevisiae)NibrinProliferating cell nuclear antigenRAD1 homolog (S. pombe)RAD17 homolog (S. pombe)RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)RAD9 homolog A (S. pombe)Retinoblastoma 1Retinoblastoma binding protein 8Retinoblastoma-like 1 (p107)Retinoblastoma-like 2 (p130)Replication protein A3, 14kDaSERTA domain containing 1S-phase kinase-associated protein 2 (p45)SMT3 suppressor of mif two 3 homolog 1 (S. cerevisiae)Transcription factor Dp-1Transcription factor Dp-2 (E2F dimerization partner 2)Tumor protein p53Ubiquitin-like modifier activating enzyme .11.12.8-1.9-1.5-2.2-1.0-1.6-1.5The gene descriptions are reproduced from the array product information from Qiagen. Greenand red shading indicate fold-changes of 2 and 2, respectively, with p 0.05. There were nogenes that did not reach the cycle threshold value within 40 cycles.
S12Table S3. Changes in expression of all genes related to inflammatory response andautoimmunity in jetPEITM or polyplex-exposed HeLa S3 cells.Fold Change in Gene ExpressionbjetPEITMGeneBankNM 001706NM 000064NM 004054NM 007293NM 006274NM 002982NM 002989NM 002990NM 002991NM 002983NM 002985NM 001295NM 005508NM 001838NM 005194NM CCL3CCL5CCR1CCR4CCR7CEBPBCSF1NM 001511CXCL1NM 001565NM 002089NM 002090NM 002994CXCL10CXCL2CXCL3CXCL5NM 002993CXCL6NM 003467NM 000639NM 001459NM 005252NM 006037NM 000572NM 000628NM 001562NM 000575NM 000576NM 000877NM 002182NM 000577NM 016584NM 000600NM 000565NM 1BIL1R1IL1RAPIL1RNIL23AIL6IL6RIL8NM 000211ITGB2NM 000595NM 002341NM 015364NM 002468LTALTBLY96MYD88NM 004555NFATC3NM 003998NFKB1NM 000625NOS2NM 000176NR3C1DescriptionaB-cell CLL/lymphoma 6Complement component 3Complement component 3a receptor 1Complement component 4A (Rodgers blood group)Chemokine (C-C motif) ligand 19Chemokine (C-C motif) ligand 2Chemokine (C-C motif) ligand 21Chemokine (C-C motif) ligand 22Chemokine (C-C motif) ligand 24Chemokine (C-C motif) ligand 3Chemokine (C-C motif) ligand 5Chemokine (C-C motif) receptor 1Chemokine (C-C motif) receptor 4Chemokine (C-C motif) receptor 7CCAAT/enhancer binding protein (C/EBP), betaColony stimulating factor 1 (macrophage)Chemokine (C-X-C motif) ligand 1 (melanoma growthstimulating activity, alpha)Chemokine (C-X-C motif) ligand 10Chemokine (C-X-C motif) ligand 2Chemokine (C-X-C motif) ligand 3Chemokine (C-X-C motif) ligand 5Chemokine (C-X-C motif) ligand 6 (granulocytechemotactic protein 2)Chemokine (C-X-C motif) receptor 4Fas ligand (TNF superfamily, member 6)Fms-related tyrosine kinase 3 ligandV-fos FBJ murine osteosarcoma viral oncogene homologHistone deacetylase 4Interleukin 10Interleukin 10 receptor, betaInterleukin 18 (interferon-gamma-inducing factor)Interleukin 1, alphaInterleukin 1, betaInterleukin 1 receptor, type IInterleukin 1 receptor accessory proteinInterleukin 1 receptor antagonistInterleukin 23, alpha subunit p19Interleukin 6 (interferon, beta 2)Interleukin 6 receptorInterleukin 8Integrin, beta 2 (complement component 3 receptor 3 and 4subunit)Lymphotoxin alpha (TNF superfamily, member 1)Lymphotoxin beta (TNF superfamily, member 3)Lymphocyte antigen 96Myeloid differentiation primary response gene (88)Nuclear factor of activated T-cells, cytoplasmic,calcineurin-dependent 3Nuclear factor of kappa light polypeptide gene enhancer inB-cells 1Nitric oxide synthase 2, inducibleNuclear receptor subfamily 3, group C, member 1(glucocorticoid receptor)GFP 2-1.12.6-6.11.41.15.4272.8-1.21.4-1.1 1084.14.5109.61.824.21.65.82.13.11.72.5-1.08.0GFP .21.12.81.18.24.07.23.5-1.21.1-1.11.9-1.11.6
S13RIPK2-1.41.2-1.12.1-1.12.2NM 001039661 TIRAP-3.8-1.0-2.4-1.1-3.4-1.5NM 003263NM 003265NM 138554NM 003268NM 006068NM 016562NM 000594NM 003807NM 17.414.63.8-1.7b2.810.84.39.6-1.31.314.12.81.2NM 003821Receptor-interacting serine-threonine kinase 2Toll-interleukin 1 receptor (TIR) domain containingadaptor proteinTLR1Toll-like receptor 1TLR3Toll-like receptor 3TLR4Toll-like receptor 4TLR5Toll-like receptor 5TLR6Toll-like receptor 6TLR7Toll-like receptor 7TNFTumor necrosis factor (TNF superfamily, member 2)TNFSF14 Tumor necrosis factor (ligand) superfamily, member 14TOLLIP Toll interacting proteinThe gene descriptions are reproduced from the array product information from Qiagen. Greenand red shading indicate fold-changes of 2 and 2, respectively, with p 0.05. Genes thatwere analyzed on the array but that did not reach the cycle threshold value within 40 cycles areCCL11, CCL13, CCL16, CCL17, CCL23, CCL4, CCL7, CCL8, CCR2, CCR3, CD40, CD40LG,CRP, CXCL9, IFNG, IL18RAP, IL1F10, IL22, IL22RA2, IL23R, IL8RA, IL8RB, IL9, KNG1, andTLR2.
S14Discussion S1. Interpretation of flow cytometry for transfection (uptake) of polyplexes intoHeLa S3 and 293A cells.For this study, it is important to quantify the fraction of the cell population that has beentransfected. In order to determine the percentage of cells that have uptaken polyplexes, weemployed polyplexes formed between jetPEITM and CX-rhodamine-labeled blank pDNA. Theexperimental details are provided above in the “pDNA Uptake Assay” and Figure S3. Wheninterpreting the data in Figure S3, two aspects are important to keep in mind: 1) the totalfluorescence shift is less than one order of magnitude and 2) the final fluorescence distributionsof the control cells and the transfected cells overlap.In the data presented in Figure S3, the entire population of HeLa S3 and 293A cellsexhibit a mean fluorescence shift of 4500 240 and 6200 390, respectively. In other words, allparts of the original fluorescence distribution of cells shift, indicating that all cells in thepopulation have uptaken the CX-rhodamine-labeled blank pDNA. This similar level of polyplexuptake across the population, at least at the level of detection of flow cytometry, is why we havetreated the cells as a single transfected population for the analyses present in this paper. Thisgeneral approach to treating the data is illustrated in the following schematic for two generalcases of homogeneous uptake of material (cases I and II) and two general cases ofinhomogeneous uptake of material (cases III and IV). The data in Figure S3 is best described bycase I.
S15All cells uptakeo equalamount of labeled DNAgiving equal x change influr escenceI# cells# cells x x not large enough to fully separatecurves thus they overlap.flur escenceflur escenceoIIAll cells uptakeo equalamount of labeled DNAgiving equal X change influr escenceo# cells X X is this case is sufficient to generate nooverlap between curves.flur escenceBoth of cases I and II show homogeneous uptake of labeled DNA bythe cell popula on, the only thing that differsis the magnitude of theoflur escence shi .A subpopula on of cellsuptakes an equal amount oflabeled DNA ( x) while theremaining cells give nouptake x# cells# cellsIIITrailing edge of fluorescence maintainsoriginal posi on as cells not uptaking DNAmaintain original distribu on offluorescence values. Again, x notsufficient to give complete separa on.flur escenceflur escenceo# cellsA subpopula on of cellsuptakes an equal amount oflabeled DNA ( X) while theremaining cells give nouptakeoIV X X is this case is sufficient to generate nooverlap between curves and twoindividual popula ons are dis nctlyobserved. Note that cells that did notuptake labeled DNA maintain originalfluorescence distribu on.flur escenceBoth of cases III and IV show inhomogeneous uptake of labeled DNAby the cell popula on, the only thing that differs is the magnitude ofthe fluorescence shi .We find it important to note that there is an alternative approach commonly used tointerpret flow cytometry data that is inappropriate for the data shown in Figure S3. Rather thanconsidering how the entire fluorescence distribution shifts, as we have described above, thissecond approach sets a cutoff defined by the fluorescence distribution of the control cells. Then,
S16for the transfected cells, uptake is deemed to occurred for all cells above this cutoff level and isdeemed to have not occurred for all cells below this cutoff limit.The two approaches outlined here result in very different interpretations regarding thefraction of the cell population transfected by polyplexes in Figure S3. In the first case, the entiredistribution is observed to shift in fluorescence and this fluorescence increase is ascribed to CXrhodamine-labeled blank pDNA uptake. Therefore, roughly 100% of the cells are assigned astransfected. In the second case, 54 3% of transfected HeLa S3 have crossed the cutoff level setby the control cells, so 54% of cells are assigned as transfected. (This figure is 68 1% for the293A cells.) Since this second method of interpreting flow cytometry data is commonly used(although often for expression experiments that give fluorescence shifts of many orders ofmagnitude, mostly similar to cases II and IV above), we found it necessary to adopt anotherapproach to determine which interpretation method was providing the correct interpretation oftransfection level for this data set.In order to distinguish between these two different approaches to interpreting the flowcytometry data, we performed a new experiment in which both flow cytometry and confocalfluorescence microscopy were employed. Confocal fluorescence microscopy allows us to see afield of cells and directly count the fraction transfected. By counting multiple fields of cells, wecan assess a fraction of uptake and compare this to the uptake values obtained by the twomethods of interpreting the flow cytometry data.For this control experiment, we again employed jetPEITM and CX-rhodamine-labeledblank pDNA to form the polyplexes. Some modifications were made to the protocol used togenerate Figure S3 in order to accommodate the inclusion of confocal fluorescence microscopyin the experiment.pDNA Uptake Measured by Confocal Microscopy and Flow CytometryCells were taken through the PKH26 procedure with no PKH26 present. The cells wereplated at 84,210 cells/well in 2-well coverglass chambers for confocal microscopy with 800 µLcomplete medium, and incubated for 5 to 6 h at 37 C with 5% CO2 for 5 hours. (The number ofcells was scaled up in comparison to the 80,000 used in 12-well plates to maintain consistency inthe number of cells per area for growth.) Cells were transfected for 3 h as described withpolyplexes formed between jetPEITM and CX-rhodamine-labeled blank pDNA according to the
S17manufacturer’s protocol and incubated for 9 h in complete medium. Two replicates were used foreach condition.CX-rhodamine-labeled blank pDNA uptake was first assessed using confocal anddifferential interference contrast (DIC) microscopy. Confocal and DIC microscopy wereperformed on live cells in complete medium using a Leica SP5X inverted microscope system(Leica Microsystems GmbH; Wetzlar, Germany). DIC was performed at 405 nm. CX-rhodaminewas excited at 575 nm (1.5 MW) and emission was measured between 595-800 nm.Immediately following the microscopy measurements, the same cell samples wereharvested and CX-rhodamine uptake was measured using flow cytometry. The cells were washedwith DPBS, harvested with trypsin, and suspended in 400 µL of DPBS. CX-rhodaminefluorescence was assessed using a BD Accuri C6 Flow Cytometer by exciting at 488 nm andmeasuring all emission greater than 670 nm.Figure S8. CX-rhodamine fluorescence intensity for representative HeLa S3 and 293A cellstreated with polyplexes formed between jetPEITM and CX-rhodamine-labeled blank pDNA. Cellswere transfected for 3 h and incubated 9 h further before being harvested and analyzed. ForHeLa S3 and 293A cells the entire population of cells exhibits a mean fluorescence shift of 1443(range of 1438 to 1448) and 1101 (1074 to 1127), respectively, consistent with a substantialdegree of transfection for all cells in the population.Once again, the entire fluorescence distribution is observed to shift with respect to thecontrol cells (analogous to case I in the above schematic). Using the first approach to analyzingthe data, we conclude that all cells have uptaken some polyplexes thus resulting in a fluorescenceshift for the entire population. In other words, roughly 100% of cells have been transfected. Inusing the second approach to analyze the data, a cutoff is set based on the control cell population
S18and we conclude that roughly 20% of the cell populations has been transfected. Clearly, the twoapproaches to data interpretation give substantially different answers. Can this be resolved byconfocal fluorescence microscopy?For the confocal fluorescence microscopy analysis, fluorescent cells were counted inmultiple fields for both 293A and HeLa S3 cells. The results are summarized in the followingtable with data given in each column for the total number of microscopy fields of viewemployed, the total number of fluorescent cells observed/total cells observed, and the percentagethis represents.Cell TypeHeLa S3293ACells only control6, 0/632, 0%3, 0/283, 0%CX-rhodamine-labledDNA only exposedcells6, 4/566, 0.7%6, 7/763, 0.1%Polyplex-ExposedCells9, 422/484, 87%8, 751/793, 95%In this case, the confocal data clearly supports the first approach to interpreting the flowcytometry data, and is in good agreement with the conclusion that almost all cells incorporatepolyplexes, thereby exhibiting a fluorescence shift. The second method of interpreting the flowcytometry data, resulting in an estimate of roughly 20% uptake, is clearly inconsistent with theconfocal microscopy analysis of the same population of cells. Examples of th
obtained with an Olympus iX70-S1F2 microscope (Olympus Corporation; Tokyo) fitted with an Olympus DP72 digital camera, and the percent of blue-green as compared to normal cells across six separate images for each sample was cou
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