Assay & Molecular Protocols

01

PEA

Proximity Extension Assay (PEA) Protocol

Ultra-sensitive protein detection using DNA-conjugated antibody pairs. Dual antibodies bind the target protein; their conjugated oligonucleotides hybridize when in proximity, enabling DNA extension and subsequent qPCR amplification. Achieves sub-pg/mL sensitivity with minimal sample volume.

4h

Total Time

<1 pg/mL

Detection Limit

96-well

Format

3

Phases

PHASE 1 Antibody Conjugation & Sample Prep (1.5h)

▼

1

Prepare DNA-Conjugated Antibodies

Conjugate capture and detection antibodies to complementary oligonucleotides using NHS-ester chemistry. Verify conjugation efficiency by SDS-PAGE (>90% expected). Optimize antibody:oligo molar ratio (typically 1:3–1:5).

NHS-PEG4-azideDBCO-oligoPBS-T

⏲ Day −1 to Day 0

2

Sample Preparation

Dilute samples in assay buffer (PBS + 0.1% BSA + 0.05% Tween-20). Prepare standard curve using recombinant protein (7-point, 4-fold serial dilution from 10 ng/mL). Include QC controls. Centrifuge lysates at 14,000×g, 10 min, 4°C.

Assay BufferRecombinant StandardsQC Controls

⏲ 30 min

3

Antibody-Sample Incubation

Add conjugated antibody pair (1–10 nM each) to sample. Incubate at RT for 1h with gentle shaking (300 rpm). Both antibodies must bind target simultaneously for proximity-dependent signal.

Ab-Oligo AAb-Oligo B

⏲ 1h

PHASE 2 DNA Extension & Amplification (2h)

▼

4

Proximity-Dependent DNA Extension

Add extension mix with T4 DNA ligase or DNA polymerase. Complementary oligo tails hybridize only when both antibodies bind the same target. Incubate 37°C, 30 min.

T4 DNA LigaseExtension BufferdNTPs

⏲ 30 min

5

qPCR Amplification

Transfer to qPCR plate. Add TaqMan master mix with target-specific primers and hydrolysis probe. Run: 95°C 10 min → 40 cycles of 95°C 15s / 60°C 1 min. Include NTC and positive controls.

TaqMan Master MixPrimersHydrolysis Probe

⏲ 1.5h

PHASE 3 Data Analysis & QC (30 min)

▼

6

Ct Value Processing

Export Ct values. Calculate ΔCt relative to extension control. Generate standard curve (4-parameter logistic fit). Interpolate sample concentrations. Flag samples with CV >15%.

GraphPad PrismPython

⏲ 20 min

7

Quality Control

Verify R² >0.99 for standard curve. Confirm QC controls within ±20%. Check NTC Ct >35. Assess inter-plate CV (<10%). Document in ELN.

BenchlingELN

⏲ 10 min

02

RT-qPCR

RT-qPCR Gene Expression Analysis Protocol

Quantitative gene expression analysis using reverse transcription and real-time PCR. Supports TaqMan probes, SYBR Green intercalating dyes, multiplex configurations, and multi-gene normalization strategies refined across 500+ validated primer pairs.

5h

Total Time

500+

Primer Pairs Validated

10 ng

Min. RNA Input

4

Phases

PHASE 1 RNA Extraction & QC (1.5h)

▼

1

Cell Lysis & RNA Extraction

Lyse cells with TRIzol (1 mL per 10 cm²) or RNeasy Mini Kit. For TRIzol: add 200 µL chloroform per mL, vortex 15s, centrifuge 12,000×g 15 min at 4°C. Transfer aqueous phase.

TRIzolChloroformRNeasy Mini Kit

⏲ 30 min

2

DNase Treatment

Treat with TURBO DNase (2U per 10 µg RNA, 37°C, 30 min). Inactivate or column cleanup. Critical for removing genomic DNA contamination.

TURBO DNaseInactivation Reagent

⏲ 45 min

3

RNA Quality Assessment

Quantify by Nanodrop: A260/280 1.8–2.1, A260/230 >1.5. Verify integrity by gel (28S:18S ~2:1) or Bioanalyzer (RIN >7.0).

NanoDropBioanalyzer

⏲ 15 min

PHASE 2 Reverse Transcription (1h)

▼

4

cDNA Synthesis

High-Capacity cDNA RT Kit: 500 ng–2 µg RNA + RT Buffer + dNTPs + Random Primers + MultiScribe RT to 20 µL. Cycle: 25°C 10 min → 37°C 120 min → 85°C 5 min → 4°C.

RT BufferdNTPsRandom PrimersMultiScribe RT

⏲ 2.5h

5

cDNA Dilution & Storage

Dilute 1:5–1:10 in nuclease-free water. Aliquot to avoid freeze-thaw. Store −20°C or −80°C. Verify with housekeeping gene (GAPDH Ct 18–22).

Nuclease-free H₂O

⏲ 10 min

PHASE 3 qPCR Setup & Run (2h)

▼

6

Primer/Probe Design

Design primers spanning exon-exon junctions (Primer3, NCBI BLAST). TaqMan probe Tm 68–70°C, primers 58–60°C. Validate with melt curve (single peak). Standard curve efficiency 90–110%.

Primer3NCBI BLASTIDT OligoAnalyzer

⏲ Validation 2h (one-time)

7

qPCR Plate Setup

Per well: 10 µL 2× TaqMan Master Mix + 1 µL 20× Assay + 4 µL cDNA + 5 µL H₂O. 384-well plate. Include NTC, NRT, inter-plate calibrator. Technical triplicates.

TaqMan Fast Advanced Master Mix384-well Plate

⏲ 30 min

8

Thermal Cycling

QuantStudio 7: 50°C 2 min → 95°C 20s → 40× (95°C 1s / 60°C 20s). SYBR: add melt curve.

QuantStudio 7

⏲ 1.5h

PHASE 4 Data Analysis (30 min)

▼

9

ΔΔCt Quantification

ΔCt = Ct(target) − Ct(geometric mean of GAPDH + ACTB + RPLP0). ΔΔCt = ΔCt(treated) − ΔCt(control). Fold change = 2^(−ΔΔCt).

QuantStudio SoftwarePrism

⏲ 20 min

10

Statistical Analysis

t-test or ANOVA (≥3 bio replicates). Report fold change ± SEM. Flag: NTC Ct <35, efficiency outside 90–110%, CV >5%.

GraphPad PrismBenchling

⏲ 10 min

03

Western

Western Blot Protein Detection Protocol

Semi-quantitative protein detection for target validation, pathway activation analysis, and expression confirmation. Supports standard and phospho-protein workflows with optimized blocking, antibody incubation, and densitometric quantification protocols.

8h

Total Time

10+

Years Experience

5 µg

Min. Protein

3

Phases

PHASE 1 Lysate Prep & Electrophoresis (3h)

▼

1

Cell Lysis

Wash 2× ice-cold PBS. RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) + protease + phosphatase inhibitors. Ice 30 min. Centrifuge 14,000×g, 15 min, 4°C.

RIPAProtease InhibitorsPhosphatase Inhibitors

⏲ 45 min

2

Protein Quantification

BCA assay, BSA standard curve (0–2 mg/mL), read 562 nm. Normalize to 1–2 µg/µL. Add 4× Laemmli + 10% β-ME. Boil 95°C 5 min.

BCA KitBSALaemmli Bufferβ-ME

⏲ 45 min

3

SDS-PAGE

Load 10–30 µg on 4–15% TGX precast gel. 80V stacking → 120V resolving (~1.5h).

TGX GelTris-Glycine-SDSMW Marker

⏲ 1.5h

PHASE 2 Transfer & Immunodetection (4.5h)

▼

4

Transfer

PVDF (methanol-activated). Wet transfer: 100V, 1h, 4°C. Large proteins >150 kDa: semi-dry 25V 30 min. Verify with Ponceau S.

PVDFTransfer BufferPonceau S

⏲ 1.5h

5

Blocking & Primary Ab

Block: 5% milk/TBS-T (total protein) or 5% BSA/TBS-T (phospho). 1h RT. Primary antibody 1:500–1:2000 overnight 4°C.

MilkBSATBS-TPrimary Ab

⏲ Overnight

6

Secondary & Detection

Wash 3× TBS-T. HRP-secondary 1:5000–1:10,000, 1h RT. Wash 4×. ECL substrate. Image on ChemiDoc.

HRP-SecondaryECLChemiDoc

⏲ 2h

PHASE 3 Quantification (1h)

▼

7

Densitometry

ImageJ/FIJI or Image Lab. Normalize to loading control. Fold change from ≥3 bio replicates.

ImageJ/FIJIPrism

⏲ 30 min

8

Stripping (Optional)

Stripping buffer (62.5 mM Tris pH 6.8, 2% SDS, 100 mM β-ME) at 50°C, 30 min. Wash 6×. Verify with ECL. Max 2–3 strip cycles.

Stripping Buffer

⏲ 45 min

04

FACS

Flow Cytometry Immunophenotyping Protocol

Multi-parametric single-cell analysis for immunophenotyping, cell sorting, and functional readouts. Supports surface and intracellular staining, multi-color panels up to 12 markers, and high-dimensional analysis with tSNE/UMAP.

4h

Sample-to-Data

12+

Panels Validated

10⁶

Events/Sample

3

Phases

PHASE 1 Sample Prep (1.5h)

▼

1

Single-Cell Suspension

Adherent: Accutase 37°C 5–10 min. Count 1–5 × 10⁶ cells per condition.

AccutaseTrypLEPBS

⏲ 20 min

2

Viability Staining

Fixable viability dye (Zombie Aqua 1:500), RT 15 min, dark. Wash with FACS buffer (PBS + 2% FBS + 1 mM EDTA).

Zombie AquaFACS Buffer

⏲ 20 min

3

Fc Block

TruStain FcX (5 µL per 10⁶ cells), ice 10 min. Don't wash.

TruStain FcX

⏲ 10 min

PHASE 2 Staining & Acquisition (2h)

▼

4

Surface Staining

Pre-titrated antibodies, ice 30 min, dark. Wash 2×. Intracellular: fix 4% PFA 15 min → perm 0.1% saponin → stain.

Fluorochrome AbsPFAPerm Buffer

⏲ 45 min

5

Compensation

Single-stain UltraComp eBeads per fluorochrome. FMO controls.

UltraComp eBeadsFMO Controls

⏲ 30 min

6

Acquisition

Resuspend 300 µL FACS buffer. FACSymphony. Min 50,000 events (live singlets). FSC-A/FSC-H doublet exclusion.

FACSymphony

⏲ 30 min

PHASE 3 Analysis (30 min)

▼

7

Gating

FlowJo: Time → FSC/SSC → Singlets → Live → Populations. FMO gates. Export stats.

FlowJo

⏲ 20 min

8

Statistics

% positive and MFI. t-test/ANOVA ≥3 replicates. Plots: histograms, dot plots, tSNE.

PrismFlowJo

⏲ 15 min

05

IP

Immunoprecipitation (IP / Co-IP) Protocol

Capture target proteins and interacting partners from cell lysates for downstream Western blot, mass spectrometry, or activity assays. Supports native IP, denaturing IP, and co-immunoprecipitation with crosslinking options.

2 days

Total Time

500 µg

Typical Input

3

Downstream Readouts

2

Phases

PHASE 1 Lysate Preparation & IP Capture (Day 1)

▼

1

Non-Denaturing Lysis

IP lysis buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA + protease/phosphatase inhibitors. 500 µL per 10⁷ cells. Rotate 4°C 30 min. Centrifuge 14,000×g, 15 min, 4°C. Save 50 µL input.

IP Lysis BufferNP-40Protease Inhibitors

⏲ 1h

2

Pre-Clear Lysate

20 µL Protein A/G beads (pre-washed) to 500 µg protein. Rotate 4°C 1h. Centrifuge 2,500×g 3 min. Transfer supernatant.

Protein A/G Agarose

⏲ 1h

3

Antibody Capture

2–5 µg capture antibody (+ IgG isotype control). Rotate overnight 4°C. Add 30 µL fresh beads, rotate 2h 4°C.

Capture AntibodyIgG ControlProtein A/G Beads

⏲ Overnight + 2h

PHASE 2 Washing, Elution & Analysis (Day 2)

▼

4

Stringent Washing

Centrifuge 2,500×g 3 min. Save flow-through. Wash 4× with high-salt buffer (300 mM NaCl). Rotate each 5 min, 4°C. Final PBS wash.

Wash BufferPBS

⏲ 45 min

5

Elution

Denaturing (WB): 2× Laemmli + β-ME, boil 5 min. Native (activity): 0.1 M glycine pH 2.5, neutralize with Tris pH 8.0.

LaemmliGlycine pH 2.5

⏲ 15 min

6

Downstream Analysis

Run IP eluate + input + IgG + flow-through on SDS-PAGE → WB. For IP-MS: on-bead trypsin digestion. Quantify enrichment >5-fold over IgG.

SDS-PAGETrypsinMass Spec

⏲ Variable

06

ELISA/MSD

Sandwich ELISA & MSD Electrochemiluminescence Protocol

Quantitative protein detection using antibody sandwich format. Supports traditional 96-well ELISA with colorimetric readout and MSD multiplex electrochemiluminescence for simultaneous quantification of up to 10 analytes per well with sub-pg/mL sensitivity.

6h

ELISA Time

8

MSD Analytes/Well

0.5 pg/mL

MSD Detection

3

Phases

PHASE 1 Coating & Blocking (3h or Pre-Coated)

▼

1

Capture Ab Coating (ELISA)

MaxiSorp plate, 1–10 µg/mL in carbonate buffer pH 9.6, 100 µL/well. Overnight 4°C or 2h 37°C. MSD: use pre-coated MULTI-SPOT plates.

MaxiSorpCarbonate BufferCapture Ab

⏲ Overnight or 2h

2

Blocking

Wash 3× PBS-T. Block 200 µL/well 1% BSA/PBS or MSD Blocker A, 1h RT shaking. Wash 3×.

BSAPBS-TMSD Blocker A

⏲ 1.5h

3

Standards & Samples

7-point 4-fold serial dilution. Blank. Cell supernatants diluted 1:2–1:10. Lysates BCA-normalized. Duplicates.

Assay DiluentRecombinant Standards

⏲ 30 min

PHASE 2 Incubation & Detection (2.5h)

▼

4

Sample Incubation

50 µL/well, 2h RT shaking or overnight 4°C. Wash 3×.

PBS-T

⏲ 2h

5

Detection Ab

Biotin-detection Ab (ELISA, 0.5–2 µg/mL) or SULFO-TAG Ab (MSD). 1h RT. Wash. ELISA: Strep-HRP 1:5000, 30 min, wash.

Biotin-Detection AbStrep-HRPSULFO-TAG Ab

⏲ 1.5h

6

Signal

ELISA: TMB substrate, develop 5–20 min dark, stop with 2N H₂SO₄, read 450 nm. MSD: 2× Read Buffer T, read on QuickPlex SQ 120.

TMBH₂SO₄MSD Read Buffer T

⏲ 30 min

PHASE 3 Data Analysis (30 min)

▼

7

Standard Curve

4PL regression. Interpolate. R² >0.99, CV <15%, QC ±20%.

MSD Discovery WorkbenchPrism

⏲ 20 min

8

Documentation

Multiplex cross-reactivity check. Lot tracking. Archive in ELN.

BenchlingELN

⏲ 10 min

07

NGS

RNA-seq Library Preparation Protocol

Sequencing-ready library preparation from extracted RNA for bulk and single-cell transcriptomics. Covers poly(A) selection, ribo-depletion, strand-specific library construction, and bioinformatics pipeline from alignment through differential expression.

8h

Library Prep

100 ng

Min. RNA Input

30+

Libraries Processed

4

Phases

PHASE 1 RNA QC & Selection (2h)

▼

1

RNA Integrity

Qubit RNA HS + Bioanalyzer/TapeStation. RIN ≥7.0 for poly(A), ≥5.0 for ribo-depletion. A260/280 1.8–2.1. Input: 100 ng–1 µg (bulk), 10k cells (10x).

QubitTapeStation

⏲ 45 min

2

mRNA Isolation

NEBNext Poly(A) module: heat 65°C 5 min, oligo-dT bead binding 2×. Degraded: RiboZero Plus.

NEBNext Poly(A)Oligo-dT BeadsRiboZero Plus

⏲ 1h

PHASE 2 Library Construction (4h)

▼

3

cDNA Synthesis

Fragment at 94°C (300 bp: 8 min). Random hexamer priming. SuperScript IV 42°C. Second strand with dUTP. AMPure XP 1.8×.

NEBNext Ultra IIAMPure XP

⏲ 1.5h

4

End Repair & Ligation

NEBNext Ultra II end repair + dA-tail. Adapter ligation 20°C 15 min. USER enzyme. Size select AMPure XP 0.6× then 0.8×.

NEBNext AdaptorUSER EnzymeAMPure XP

⏲ 1.5h

5

PCR Enrichment

Q5 Master Mix + i7/i5 dual index primers. 98°C 30s → 8–12× (98°C 10s / 65°C 75s) → 65°C 5 min. AMPure XP 1×.

Q5 Master MixIndex Primers

⏲ 1h

PHASE 3 QC & Pooling (1.5h)

▼

6

Library QC

Qubit dsDNA HS + TapeStation. Expected peak 350–450 bp. nM = (ng/µL × 10⁶)/(660 × size). Re-purify if adapter dimer present.

QubitTapeStation D1000

⏲ 45 min

7

Pooling

Equimolar 4 nM. Denature 0.2N NaOH. Load per platform specs. 1% PhiX. Submit to core/CRO.

NaOHPhiXNovaSeq

⏲ 30 min

PHASE 4 Bioinformatics

▼

8

Alignment

FastQC → Trim Galore → STAR (GRCh38, 2-pass) → featureCounts. 10x: CellRanger → Seurat.

STARfeatureCountsCellRanger

⏲ 2–4h compute

9

DE & Pathway Analysis

DESeq2 (bulk) or SCTransform (SC). DE: padj <0.05, |log2FC| >1. GSEA MSigDB. GO/KEGG with clusterProfiler. Volcano, heatmaps, UMAP.

DESeq2SeuratclusterProfiler

⏲ Variable

08

Culture

Mammalian Cell Culture & iPSC Maintenance Protocol

Routine maintenance protocols covering iPSC expansion and QC, immortalized cell line culture (HEK293, CHO, Vero E6), cryopreservation, mycoplasma testing, and cell health documentation across BSL-1 through BSL-2+ environments.

15+

Cell Types

10+

Years Experience

5L

Bioreactor Scale

3

BSL Levels

PHASE 1 iPSC Maintenance

▼

1

Daily Feeding

Pre-warmed mTeSR Plus (2 mL/well 6-well). Phase microscopy: compact colonies, <5% differentiation. Remove differentiated regions. Feed daily.

mTeSR PlusMatrigel

⏲ 15 min/plate

2

Passaging

At 70–80% confluency (every 4–5 days). PBS wash. GCDR 37°C 6–8 min. mTeSR Plus + 10 µM Y-27632. Triturate to small clumps. Split 1:6–1:12 on Matrigel. Remove ROCK inhibitor after 24h.

GCDRY-27632Matrigel

⏲ 30 min

3

Cryopreservation

Small clumps in mFreSR (1 mL/vial, 1–2 × 10⁶ cells). CoolCell at −80°C overnight → LN₂ within 48h. Record passage, date, line ID, myco status.

mFreSRCoolCellLN₂

⏲ 30 min

PHASE 2 Immortalized Lines

▼

4

Routine Maintenance

DMEM + 10% FBS + Pen/Strep (HEK293). 0.05% Trypsin-EDTA 3 min 37°C. Split 1:8–1:12. Monitor doubling (HEK293 ~24h).

DMEMFBSTrypsin-EDTA

⏲ 20 min

5

Mycoplasma & Authentication

MycoAlert every 2 weeks. Positive: Plasmocin 25 µg/mL 2 weeks. STR profiling on thaw and every 20 passages.

MycoAlertPlasmocinSTR

⏲ 30 min

PHASE 3 QC & Documentation

▼

6

Health Monitoring

Trypan blue viability >95%. Daily morphology. Growth curves. Discard if viability <90%, abnormal morphology, passage >30 (iPSC) or >50 (immortalized), myco+.

Trypan BlueHemocytometer

⏲ 10 min

7

Documentation

Benchling entries for every passage/freeze/thaw. Lot tracking. LN₂ inventory monthly. Chain-of-custody for patient iPSCs. Karyotyping every 10 passages.

BenchlingG-banding

⏲ 15 min

09

qPCR Data

RT-qPCR Gene Expression Data

Representative RT-qPCR datasets from iPSC-derived neuronal, cardiac, and pancreatic differentiation experiments. Expression is normalized to geometric mean of housekeeping genes (GAPDH, ACTB, RPLP0) and presented as fold change (2^−ΔΔCt) relative to undifferentiated iPSCs. Error bars represent SEM from n=3 biological replicates. All primer pairs validated with efficiency 90–110% and single melt-curve peaks. View full qPCR data on the Genetics & Molecular Biology page →

500+

Primer Pairs

ΔΔCt

Quantification

3

Bio Replicates

384

Well Format

DATA iPSC-Derived Dopaminergic Neuron Markers (DIV 42)

▼

Fold change vs undifferentiated iPSCs. GAPDH/ACTB/RPLP0 normalization. TaqMan probes. QuantStudio 7. n=3 biological replicates per gene. *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA with Dunnett's post-hoc).

TH (142.3×)NURR1 (98.7×)FOXA2 (87.4×)LMX1A (63.1×)EN1 (54.8×)PITX3 (41.2×)DAT (28.6×)GIRK2 (19.4×)

DATA Multi-Lineage Differentiation Heatmap

▼

Log₂(fold change) relative to undifferentiated iPSCs. Red = upregulated, blue = downregulated. Rows = genes, columns = lineages. RT-qPCR with TaqMan probes, GAPDH normalization, n=3.

DATA Host Gene Expression After SARS-CoV-2 Spike Exposure

▼

RT-qPCR profiling of host endocytic and innate immunity genes in Calu-3 cells after 6h exposure to purified His₆-tagged SARS-CoV-2 Spike (10 µg/mL). Fold change vs vehicle control. GAPDH/ACTB normalization. n=3.

ACE2TMPRSS2CLTCPIK3C3PIKFYVEIFNB1IFIT1MX1

10

Flow Data

Flow Cytometry Quantitative Data

Multicolor flow cytometry datasets from immunophenotyping experiments across immune cell profiling (lymph node and spleen tissue), iPSC differentiation QC, and cancer immunology co-cultures. Acquired on BD LSRFortessa and Cytek Aurora platforms. Analyzed in FlowJo v10 with tSNE/UMAP dimensionality reduction. All data gated on live singlets (Zombie Aqua⁻, FSC-A/FSC-H). View full flow data on the Genetics & Molecular Biology page →

12+

Panels Validated

50k+

Events/Sample

BD / Cytek

Platforms

FlowJo

Analysis

DATA Lymph Node Immune Cell Frequencies — WT vs LRRK2 G2019S

▼

Immune cell subset frequencies in cervical lymph nodes of WT and LRRK2 G2019S knock-in mice (12 months, n=8 per genotype). Surface panel: CD45, CD3, CD4, CD8, CD19, NK1.1, CD11b, Ly6G, Ly6C. Intracellular: Ki67, FoxP3. BD LSRFortessa acquisition. *p<0.05, **p<0.01 (unpaired t-test).

CD4+ T cellsCD8+ T cellsB cells (CD19+)NK cellsTregs (FoxP3+)MonocytesNeutrophils

DATA iPSC Differentiation QC — Multi-Lineage Marker Expression

▼

Flow cytometry assessment of lineage-specific markers at terminal differentiation. Dopaminergic neurons (DIV 42): TH, FOXA2, TUJ1. Cardiomyocytes (Day 30): cTnT, MYH7, NKX2.5. Pancreatic β-cells (Day 28): C-Peptide, NKX6.1, PDX1. n=3 independent differentiations per lineage.

DATA T-Cell Checkpoint Expression — PB vs TIL

▼

Surface checkpoint receptor expression on CD3+CD8+ T cells from peripheral blood (PB) vs tumor-infiltrating lymphocytes (TIL). Markers: PD-1, CTLA-4, TIM-3, LAG-3, TIGIT. Multicolor flow cytometry, Cytek Aurora. MFI normalized to isotype controls. n=6 donors.

11

Virology

Virology — Viral Internalization & Transduction

Quantitative virology datasets spanning SARS-CoV-2 spike internalization via clathrin-mediated endocytosis (CME), pseudoviral infectivity assays, and viral vector transduction optimization. This work led to the first paper demonstrating the cellular entry mechanism of SARS-CoV-2 (J. Biol. Chem. 2021; 500+ citations) and contributed to host-directed antiviral strategies targeting PIKfyve and CSNK2 kinases in collaboration with UNC Chapel Hill, GSK, and the Structural Genomics Consortium. View full virology data on the Genetics & Molecular Biology page →

500+

Citations (JBC 2021)

4

Cell Lines Validated

BSL-2/3

Biosafety

3

Viral Vector Systems

DATA SARS-CoV-2 Spike Internalization Kinetics

▼

Time course of His₆-tagged SARS-CoV-2 Spike glycoprotein internalization in HEK-293T-ACE2 cells. Cells were pulsed with Spike (5 µg/mL, 4°C, 30 min) then chased at 37°C. Intracellular Spike was quantified by confocal microscopy (anti-His₆ AF488) and normalized to total bound Spike at t=0. Pharmacological conditions: DMSO vehicle, Pitstop 2 (30 µM, CME inhibitor), Dynasore (80 µM, dynamin inhibitor), CHC siRNA (72h, 95% knockdown). n=3, 50+ cells scored per condition per time point.

HEK-293T-ACE2Spike-His₆Pitstop 2DynasoreCHC siRNAConfocal

DATA CME-Dependent Spike Uptake & Pseudoviral Infectivity

▼

Quantification of SARS-CoV-2 Spike uptake (% of DMSO control) and pseudoviral infectivity (GFP+ cells, %) across four cell lines under CME disruption. CHC siRNA: 72h, 95% knockdown. Pitstop 2: 30 µM, 1h pre-treatment. Dynasore: 80 µM, 1h pre-treatment. Pseudovirus: Spike-pseudotyped lentivirus, 48h infection. n=3.

HEK-293T-ACE2VeroCalu-3Caco-2PseudovirusGFP Quantification

DATA Host Kinase Inhibitor Dose-Response — Viral Entry & Replication

▼

Dose-response curves for PIKfyve and CSNK2 inhibitors on SARS-CoV-2 pseudoviral entry (GFP+, 48h) and viral replication (RNA copy number, 72h) in Calu-3 cells. Compound 17 (PIKfyve probe, IC₅₀ = 0.82 nM): matched with negative control Compound 30. Silmitasertib (CSNK2 clinical-grade inhibitor). Apilimod (PIKfyve, clinical). n=4, technical triplicates.

Compound 17 (PIKfyve)Apilimod (PIKfyve)Silmitasertib (CSNK2)SGC-CK2-1 (CSNK2)Calu-3

DATA Viral Transduction Efficiency — Lentivirus, Adenovirus & AAV

▼

Transduction efficiency of three viral vector systems across iPSC-derived cell types at optimized MOIs. Lentivirus (VSV-G pseudotyped, 3rd-generation): shRNA-LAMP2 and reporter constructs. Adenovirus (Ad5, CMV promoter): α-synuclein-HA overexpression. AAV (serotypes 1, 2, 6, 9): GFP reporter for tropism mapping. Efficiency measured by flow cytometry (GFP+ or tag+) and Western blot at 72h (lenti/Ad5) or 7 days (AAV) post-transduction. n=3.

Lentivirus (VSV-G)Adenovirus (Ad5)AAV1/2/6/9iPSC-DA NeuronsiPSC-CardiomyocytesHEK-293T

DATA Lentiviral MOI Optimization — Efficiency vs Viability

▼

Dual-axis optimization of lentiviral transduction efficiency (GFP+ by flow cytometry) and cell viability (trypan blue exclusion) across MOI range in iPSC-derived dopaminergic neurons (DIV 35). VSV-G pseudotyped, 3rd-generation lentivirus with pLKO.1-shLAMP2-GFP construct. Polybrene (8 µg/mL) supplementation. 72h post-transduction. n=3.

MOI 1–50PolybreneGFP+ FlowViabilityshLAMP2

Proximity Extension Assay (PEA) Protocol

PHASE 1 Antibody Conjugation & Sample Prep (1.5h)

PHASE 2 DNA Extension & Amplification (2h)

PHASE 3 Data Analysis & QC (30 min)

RT-qPCR Gene Expression Analysis Protocol

PHASE 1 RNA Extraction & QC (1.5h)

PHASE 2 Reverse Transcription (1h)

PHASE 3 qPCR Setup & Run (2h)

PHASE 4 Data Analysis (30 min)

Western Blot Protein Detection Protocol

PHASE 1 Lysate Prep & Electrophoresis (3h)

PHASE 2 Transfer & Immunodetection (4.5h)

PHASE 3 Quantification (1h)

Flow Cytometry Immunophenotyping Protocol

PHASE 1 Sample Prep (1.5h)

PHASE 2 Staining & Acquisition (2h)

PHASE 3 Analysis (30 min)

Immunoprecipitation (IP / Co-IP) Protocol

PHASE 1 Lysate Preparation & IP Capture (Day 1)

PHASE 2 Washing, Elution & Analysis (Day 2)

Sandwich ELISA & MSD Electrochemiluminescence Protocol

PHASE 1 Coating & Blocking (3h or Pre-Coated)

PHASE 2 Incubation & Detection (2.5h)

PHASE 3 Data Analysis (30 min)

RNA-seq Library Preparation Protocol

PHASE 1 RNA QC & Selection (2h)

PHASE 2 Library Construction (4h)

PHASE 3 QC & Pooling (1.5h)

PHASE 4 Bioinformatics

Mammalian Cell Culture & iPSC Maintenance Protocol

PHASE 1 iPSC Maintenance

PHASE 2 Immortalized Lines

PHASE 3 QC & Documentation

RT-qPCR Gene Expression Data

DATA iPSC-Derived Dopaminergic Neuron Markers (DIV 42)

DATA Multi-Lineage Differentiation Heatmap

DATA Host Gene Expression After SARS-CoV-2 Spike Exposure

Flow Cytometry Quantitative Data

DATA Lymph Node Immune Cell Frequencies — WT vs LRRK2 G2019S

DATA iPSC Differentiation QC — Multi-Lineage Marker Expression

DATA T-Cell Checkpoint Expression — PB vs TIL

Virology — Viral Internalization & Transduction

DATA SARS-CoV-2 Spike Internalization Kinetics

DATA CME-Dependent Spike Uptake & Pseudoviral Infectivity

DATA Host Kinase Inhibitor Dose-Response — Viral Entry & Replication

DATA Viral Transduction Efficiency — Lentivirus, Adenovirus & AAV

DATA Lentiviral MOI Optimization — Efficiency vs Viability

Assay Protocol Development Lifecycle