Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System

Summary

This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E^-/- HeLa cells using the CRISPR/Cas9 system.

Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors.

In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

Introduction

Engineered genome editing mediates the targeted modifications of genes in a variety of cells and organisms. In eukaryotes, site-specific mutagenesis can be introduced by the applications of sequence-specific nucleases that stimulate homologous recombination of target DNA¹. In recent years, several genome editing technologies, including zinc finger nucleases (ZFNs)^2,3, transcription activator-like effector nucleases (TALENs)^4,5, and homing meganucleases^6,7, have been engineered to cleave genomes at specific sites, but these approaches require complex protein engineering and redundant experimental procedures. Studies have shown that the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an efficient gene editing technology, which specifically mediates RNA-guided, site-specific DNA cleavage in a wide variety of cells and species^8,9,10,11. The CRISPR/Cas9 gene knockout technology has revolutionized the fields of basic biology, biotechnology, and medicine¹².

Bacteria and most archaea have evolved an RNA-based adaptive immune system that uses CRISPR and Cas proteins to identify and destroy viruses and plasmids¹³. Streptococcus pyogenes Cas9 (SpCas9) endonuclease contains the RuvC-like Holliday junction resolvase (RuvC) and His-Asn-His (HNH) domain, which can efficiently mediate sequence-specific, double-stranded breaks (DSBs) by providing a synthetic single-guide RNA (sgRNA) containing CRISPR RNAs (crRNA) and trans-activating crRNA (tracrRNA)^14,15,16. DSBs can be repaired through the indel-forming non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway, which introduces multiple mutations, including insertions, deletions, or scar-less single nucleotide substitutions, in mammalian cells^1,8. Both the error-prone NHEJ and the high-fidelity HDR pathway can be used to mediate gene knockout through insertions or deletions, which can cause frameshift mutations and premature stop codons¹⁰.

Kinesin-7 CENP-E is required for kinetochore-microtubule attachment and chromosome alignment during cell division^17,18,19. Antibody microinjection^20,21, siRNA depletion^22,23, chemical inhibition^24,25,26, and genetic deletion^27,28,29 of CENP-E leads to chromosome misalignment, the activation of spindle assembly checkpoint and mitotic defects, which results in aneuploidy and chromosomal instability^19,30. In mice, CENP-E deletion results in abnormal development and embryonic lethality at the very early stages of development^27,29,31. Genetic deletion of CENP-E usually leads to chromosome misalignment and cell death^26,27,29, which is an obstacle in studying the functions and mechanisms of the CENP-E proteins.

A recent study has established a conditional CENP-E knockout cell line using an Auxin-inducible CRISPR/Cas9 gene-editing method³², which enables rapid degradation of CENP-E proteins in a relatively short time³³. However, to date, stable CENP-E knockout cell lines have not been established, which is an unresolved technical challenge in CENP-E biology. Considering genetic robustness³⁴, genetic compensation responses^35,36,37, and complex intracellular environments, as the direct consequences of complete deletion of CENP-E may be complex and unpredictable, it is important to establish CENP-E knockout cell lines for the investigation of mechanisms of chromosome alignment, spindle assembly checkpoint, and downstream signaling pathways.

The discovery and applications of CENP-E inhibitors are important for cancer treatment. To date, seven types of CENP-E inhibitors have been found and synthesized, including GSK923295 and its derivatives^24,25, PF-2771^38,39, imidazo[1,2-a]pyridine scaffold derivatives^40,41, compound-A^42,43, syntelin^44,45, UA62784⁴⁶, and benzo[d]pyrrolo[2,1-b]thiazole derivatives⁴⁷. Among these inhibitors, GSK923295 is an allosteric and efficient CENP-E inhibitor that binds to the motor domain of CENP-E and inhibits CENP-E microtubule-stimulated ATPase activity with a K_i of 3.2 ± 0.2 nM^24,25. However, compared with the inhibitory effects of GSK923295 on cultured cancer cells, the therapeutic effects of GSK923295 in clinical cancer patients are not ideal^48,49, which also raised concerns about the specificity of GSK923295 for CENP-E. Moreover, the specificity and side effects of other CENP-E inhibitors on the CENP-E proteins are key issues in cancer research.

In this study, we have completely knocked out the CENP-E gene in HeLa cells using the CRISPR/Cas9 system. Three optimized phenotype-based screening strategies have been established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, to improve the screening efficiency and success rate of CENP-E gene editing. Furthermore, CENP-E knockout cell lines can be used to test the specificity of candidate compounds for CENP-E.

Protocol

1. Construction of the CRISPR/Cas9 gene knockout vectors

1. Select the target genomic DNA sequence on the human CENP-E gene (GenBank Accession No. NM_001286734.2) and design the sgRNA using an online CRISPR design tool (http://crispor.tefor.net/).
2. Input a single genomic sequence, select the genome of "Homo sapiens-human-UCSC Dec 2013 (hg38 analysis set) + single nucleotide polymorphisms (SNPs): dbSNP148", and select the protospacer adjacent motif "20 bp-NGG-spCas9". Choose two guide sequences, including sgRNA-1, 5'-CGGCCGCACTCGCACGCAGA-3' and sgRNA-2 5'-TTCTTTAGAGACGCGGGCTC-3', according to the specificity scores⁵⁰, the predicted efficiency¹⁰, and the minimal off-targets.
3. Order and synthesize the single-stranded DNA oligonucleotides (ssODNs). Dilute the ssODNs in ddH₂O to a final concentration of 100 µM. To phosphorylate and anneal the sgRNA oligos, add 1 µL of forward oligo, 1 µL of reverse oligo, 0.5 µL of T4 polynucleotide kinase, 1 µL of T4 polynucleotide kinase buffer, and 6.5 µL of RNase-free water in a polymerase chain reaction (PCR) tube, and incubate the tube at 37 °C for 30 min, 95 °C for 5 min; then, ramp down to 25 °C at 5 °C min^-1.
4. Digest the pX458 plasmid using the Bbs restriction enzyme. Add 1 µg of the pX458 plasmid, 2 µL of 10x buffer G, 1 µL of Bbs, and add RNase-free ddH₂O to 20 µL in a 1.5 mL centrifuge tube. Incubate the tube at 37 °C for 2 h. Then, purify the linear plasmid using the column DNA gel extraction kit according to the manufacturer's protocols.
5. Ligate the sgRNA oligos into the pX458 plasmid (pSpCas9(BB)-2A-green fluorescent protein (GFP) plasmid, Addgene ID. 48138). Add 1 µL of the annealed oligonucleotides, 25 ng of the linear plasmid, 1 µL of 10x T4 DNA ligation buffer, 0.5 µL of T4 DNA ligase (350 U/µL) and make up to 10 µL with ddH₂O in a 1.5 mL centrifuge tube. Incubate the ligation solution at 16 °C for 2 h.
6. Incubate 10 µL of the constructed plasmid with the competent DH5α cells on ice for 30 min, heat shock at 42 °C for 45 s, and immediately place them on ice for 5 min. Add 500 µL of Luria-Bertani (LB) medium and seed the cells on an LB plate containing ampicillin (100 µg/mL). Incubate it at 37 °C for 16 h and screen the resistant clones.
7. Select 5-10 single colonies with a sterilized pipette tip and transfer them to a 1 mL culture of LB medium with ampicillin (100 µg/mL). Incubate the culture at 37 °C and shake it at 180 rpm for 12-16 h.
8. Isolate the plasmid DNA using a plasmid extraction kit according to the manufacturer's protocols. Validate the plasmid DNA of each colony by Sanger sequencing from the U6 promoter site using the U6-Fwd primer 5'-GAGGGCCTATTTCCCATGATTCC-3'.
9. Extract and purify the plasmids using the endo-free plasmid DNA kit according to the manufacturer's protocols.

2. Transfection, isolation, and screening of the CENP-E knockout HeLa cells

1. Culture HeLa cells in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO₂.
2. Incubate the HeLa cells using 0.25% trypsin/ethylene diamine tetraacetic acid (EDTA) at 37 °C for 2-3 min, dissociate the cells by pipetting gently, and then, seed the cells in new plates at a dilution ratio of 1:4 for cell passage.
3. Seed the cells in a 12-well plate, and culture to 60-70% confluency before transfection. Transfect the pX458-sgRNA plasmids into HeLa cells using the referenced reagents according to the manufacturer's protocols (see the Table of Materials).
   1. Gently mix 1 µg of validated pX458-sgRNA plasmid and 50 µL of reduced serum medium in tube A. Then, gently mix 2 µL of the transfection reagent and 50 µL of reduced serum medium in tube B. Incubate tubes A and B separately at room temperature for 5 min.
   2. Mix both tube A and tube B gently and incubate at room temperature for 5 min. Add the mixture to a 12-well plate, incubate the cells for 6 h, and replace the medium with a fresh DMEM medium.
4. Examine HeLa cells after 24 h or 48 h using a fluorescence microscope for transfection efficiency. After transfection for 48 h, completely dissociate the transfected HeLa cells using 0.25% trypsin/EDTA at 37 °C for 3-5 min, count the number of cells using a Neubauer chamber or an automated cell counter, and plate the cells into three separate 96-well plates for each transfected population according to serial dilution methods¹⁰.
5. Return the cells to a humidified incubator and then culture them for another 1-2 weeks. Change the culture medium once every 3 days. For the phenotype-based screening and validation of CENP-E knockout, dissociate and expand the cells in 24-well or 12-well plates for 5-7 days.
6. Screen the mutant cells with smaller cell colony diameters using an inverted microscope equipped with 10x and 20x objective lenses.
   NOTE: CENP-E mutations usually result in a significant increase in the number of dividing cells in cell colonies, which can be one of the key indicators for screening the CENP-E mutant cells.
7. Harvest the cells for DNA extraction using the column animal genomic DNA extraction kit according to the manufacturer's protocols. Set up the PCR reactions as follows: 0.25 µL of Taq polymerase, 5 µL of 10x buffer (Mg²⁺ plus), 4 µL of dNTP mixtures, 500 ng of DNA template, 1 µL of forward oligo, 1 µL of reverse oligo, and adjust ddH₂O to 50 µL. Use the following PCR program settings for gene amplification: 98 °C, 10 s; 98 °C, 10 s, 55 °C, 30 s, 72 °C, 60 s for 33 cycles; 72 °C, 10 min, and then hold at 4 °C.
   NOTE: The specific primers for cloning of the target locus are listed as follows: CENP-E target F1, 5'-GAGGGTCCTGGCCATTTTCCTG-3'; CENP-E target R1, 5'-AGATCTCCGATCCTCCCCTGTC-3'; CENP-E target F2, 5'-TGGTAACTGCATTTTGGTGTTCTAC-3'; CENP-E target R2, 5'-CCTGTTGCAACGTGAGGGAAG-3'.
8. Ligate the target DNA into the pMD18-T vector according to the manufacturer's protocols. Transfect the ligated plasmid into competent DH5α cells and culture for selection of clones.
9. Perform Sanger sequencing to determine the types of CENP-E knockout. Isolate the plasmid DNA using a plasmid extraction kit according to the manufacturer's protocols. Carry out Sanger sequencing of the plasmid DNA of each colony using the M13 forward and reverse primers. M13F primer, 5'-TGTAAAACGACGGCCAGT-3'; M13R primer, 5'-CAGGAAACAGCTATGACC-3'.
10. Seed the wild-type and CENP-E mutant HeLa cells on 12 mm glass coverslips in a 24-well plate, respectively. Remove the complete DMEM medium and fix the cells in 4% paraformaldehyde/phosphate-buffered saline (PBS) fixative solution at room temperature for 10 min.
11. Stain the nuclei with the 4',6-diamidino-2-phenylindole (DAPI) solution at room temperature for 5 min. Screen and validate the CENP-E mutant cells based on the phenotype of chromosome alignment using a fluorescence microscope equipped with a Plan Fluor 40x/ Numerical Aperture (NA) 0.75 objective.
12. Screen and validate the CENP-E knockout cells using immunofluorescence staining and analysis (see section 3) of CENP-E proteins.

3. Immunofluorescence staining and high-resolution confocal microscopy

1. Fix the cells with 4% paraformaldehyde/PBS fixative solution at room temperature for 10 min and immerse for 2 x 5 min in 1x PBS.
   NOTE: Ensure that the cells are in healthy condition and cells are collected and fixed gently to avoid metaphase cell detachment.
2. Permeabilize the cells with 0.25% Triton X-100/PBS at room temperature for 10 min and immerse them in 1x PBS for 2 x 5 min.
3. Block the cells with 1% BSA/PBST (0.1% Tween 20 in PBS) at room temperature for 1 h. Dilute the primary antibodies with 1% BSA/PBST and incubate the samples at 4 °C for 12 h.
4. Discard the primary antibody solutions, rinse the cells 3 x 5 min with 1x PBS, and incubate the cells with diluted secondary antibodies at room temperature for 2 h.
5. Discard the secondary antibodies and rinse the cells in 1x PBS for 3 x 5 min.
6. Stain the nuclei with DAPI at room temperature for 5 min. Mount the slides with the mounting medium. Seal the slides with nail polish.
7. Observe and record the fluorescence images using a scanning confocal microscope equipped with a 63x/NA 1.40 objective.

4. Chromosome preparation and karyotype analysis

1. Culture the wild-type and CENP-E^-/- HeLa cells in complete DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin for 24 h and then, incubate the wild-type and CENP-E^-/- HeLa cells with 300 nM colchicine for 5 h.
2. Incubate the cells with 0.25% trypsin/EDTA at 37 °C for 3 min and collect the cells into 1.5 mL centrifuge tubes.
3. Centrifuge the cells at 1,000 × g for 5 min at room temperature, discard the supernatants, add 1.2 mL of 0.075 mol/L KCl solution, and incubate at 37 °C for 20 min.
4. Centrifuge the cells at 1,000 × g for 5 min at room temperature, discard the supernatants, add 0.2 mL of the fixative solution (methanol: glacial acetic acid = 3:1) for prefixation, and mix gently for 1 min.
5. Centrifuge the cells at 1,000 × g for 5 min at room temperature, collect the pellets, add 1.5 mL of the fixative solution at room temperature for 10 min, and then centrifuge at 1,000 × g for 5 min at room temperature.
6. Discard the supernatants, add 0.6 mL of the fixative solutions, and prepare a cell suspension. Add 3-5 drops of the cell suspensions from a height of 35-40 cm on the ice slides.
   NOTE: Keep the height for releasing the cell suspensions onto the slides at 35-40 cm; otherwise, the chromosomes may be too dispersed or not separated from each other.
7. Dry the slides immediately with an alcohol lamp, stain the samples with 10% Giemsa staining solution for 7 min, rinse the slides with running water for 2 min, and observe the samples using a light microscope equipped with a Plan Fluor 40x/NA 0.75 objective.

5. Cell colony formation assay

1. Prepare the cell suspensions in the 6-well plates at a cell density of 1,000-2,000 cells/well. Gently shake the plates to evenly distribute the cells.
2. Place the 6-well plates into a CO₂ incubator and incubate for 2-3 weeks at 37 °C with 5% CO₂. Change culture medium once every 5 days. Record the images using an inverted microscope equipped with 10x and 20x objective lenses. Harvest the cells when colonies are visible (hundreds of cells within a clone).
3. To study the effects of GSK923295, culture the cells in the 6-well plates for 24 h, and add 2 mL of GSK923295 at the final concentrations of 10, 25, 50, 100, and 200 nM.
   NOTE: Do not move the culture dish at the early stage of clone formation to avoid cell shedding, which can form new clones and affect the screening of transfected cells.
4. Discard the culture medium and fix the cells with 1% PFA at room temperature for 10 min. Stain the colonies with 0.1% crystal violet staining solution at room temperature for 15 min. Rinse the samples with 1x PBS three times. Record the images of cell colonies and quantify the diameters of each colony using ImageJ software. Choose the Straight line selection tool, click Analyze, choose Measure, and record the length.
5. Rinse the colonies with 1 mL of 10% acetic acid solution for 5 min. Transfer 200 µL of the supernatant solutions in a 96-well plate, and measure absorbance values using a microplate spectrophotometer at A_{600 nm}.

6. Cell viability assay

1. Seed the cells in the 24-well plates and culture the cells for 48 h to 80-90% density.
2. Incubate the cells with 100 µL of 0.25% trypsin/EDTA at 37 °C for 3 min.
3. Mix 400 µL of PBS with the cells with a pipette gun. Transfer 100 µL of the cell suspensions into the 96-well plate.
4. Add 20 µL of MTS solution in each well with a final concentration of 317 µg/mL and mix gently according to the manufacturer's protocols. Incubate the samples at 37 °C for 1-3 h in a CO₂ incubator.
5. Record the absorbance values of each well at the absorbance peak of A_{490 nm} using a microplate spectrophotometer. Use a reference wavelength of A_{630 nm} to subtract the background contributed by cell debris and nonspecific absorbance (Cell viability = A_490nm- A_{630 nm}).

Results

The CENP-E^-/- HeLa cells were successfully generated using the CRISPR/Cas9 system (Figure 1). The timeline and critical experimental steps of this method are shown in Figure 1. First, we designed and synthesized the CENP-E-specific sgRNAs, annealed and ligated the sgRNAs into the pX458 plasmid, transfected the plasmid into HeLa cells, and cultured them for 48 h. The transfected cells were dissociated and seeded in a 96-well plate usi...

Discussion

Kinesin-7 CENP-E is a key regulator in chromosome alignment and spindle assembly checkpoint during cell division^17,19,20. Genetic deletion of CENP-E usually results in the activation of spindle assembly checkpoint, cell cycle arrest, and cell death^27,29,51,52. Thus, the construction of stable CENP-...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Gibco	25200056
1.5 mL centrifuge tube	Axygen	MCT-150-C
24-well plate	Corning	3524
4S Gelred, 10,000x in water	Sangon Biotech (Shanghai)	A616697
50 mL centrifuge tube	Corning	430828
6 cm Petri dish	Corning	430166
95% ethanol	Sinopharm Chemical Reagent	10009164
96-well plate	Corning	3599
Acetic acid	Sinopharm Chemical Reagent	10000218	Dissolve in H2O to prepare a 10% working solution.
Agarose	Sangon Biotech (Shanghai)	A620014
Alexa Fluor 488-labeled Goat Anti-Mouse IgG(H+L)	Beyotime	A0428	For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.
Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H+L)	Beyotime	A0423	For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.
Alexa Fluor 555-labeled Donkey Anti-Mouse IgG(H+L)	Beyotime	A0460	For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.
Anhydrous ethanol	Sinopharm Chemical Reagent	100092690
Anti-BubR1 rabbit monoclonal antibody	Abcam	ab254326	For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution
Anti-CENP-B mouse monoclonal antibody	Santa Cruz Biotechnology	sc-376392	For immunofluorescence. Dissolve in 1% BSA/PBST. 1:50 dilution.
Anti-CENP-E rabbit monoclonal antibody	Abcam	ab133583	For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution.
Anti-fade mounting medium	Beyotime	P0131	Slowing down the quenching of fluorescent signals.
Anti-α-tubulin mouse monoclonal antibody	Abcam	ab7291	For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution.
Biotek Epoch Microplate Spectrophotometer	Biotek Instruments	Biotek Epoch
Bovine Serum Albumin (BSA)	Sinopharm Chemical Reagent	69003435
BpiI (BbsI)	Thermo Fisher Scientific	ER1011
CellTiter 96 aqueous one solution cell proliferation assay	Promega	G3580
Centrifuge	Eppendorf	5424BK745380
Colchicine	Sinopharm Chemical Reagent	61001563
Confocal scanning microscope	Leica	Leica TCS SP8
Coverslip	CITOTEST	80344-1220
DAPI	Beyotime	C1006
DH5α competent cells	Sangon Biotech (Shanghai)	B528413
DL2000 DNA marker	TaKaRa	3427A
Dulbecco's Modified Eagle Medium (DMEM)	Gibco	C11995500BT
Endo-free plasmid mini kit	Omega	D6950
Ezup Column Animal Genomic DNA Purification Kit	Sangon Biotech (Shanghai)	B518251
Fetal bovine serum	Zhejiang Tianhang Biotechnology	11011-8611
Gentian violet	Sinopharm Chemical Reagent	71019944	Dissolve in PBS to prepare 0.1% gentian violet/PBS.
Giemsa staining solution	Sinopharm Chemical Reagent	71020260
GraphPad Prism version 8.0 software	GraphPad	www.graphpad.com	Statistical analysis.
GSK923295	MedChemExpress	HY-10299
HeLa cell line	ATCC	CCL-2
Humidified incubator	Heal Force	HF90/HF240
Image J software	National Institutes of Health	https://imagej.nih.gov/ij/	Image processing and analysis.
Inverted microscope	Nanjing Jiangnan Novel Optics	XD-202
LB agar powder	Sangon Biotech (Shanghai)	A507003
Lipo6000 transfection reagent	Beyotime	C0526
Nikon Ti-S2 microscope	Nikon	Ti-S2
Opti-MEM reduced serum medium	Gibco	31985070
Paraformaldehyde	Sinopharm Chemical Reagent	80096618	Dissolve in PBS to prepare 4% paraformaldehyde/PBS.
Penicillin-streptomycin solution	HyClone	SV30010
SanPrep column DNA gel extraction kit	Sangon Biotech (Shanghai)	B518131
SanPrep column plasmid mini-preps kit	Sangon Biotech (Shanghai)	B518191
T4 DNA ligase	TaKaRa	2011A
T4 polynucleotide kinase	TaKaRa	2021A
TaKaRa Ex Taq	TaKaRa	RR001A
Triton X-100	Sinopharm Chemical Reagent	30188928	Dissolve in PBS to prepare 0.25% Triton X-100/PBS.
Tween 20	Sinopharm Chemical Reagent	30189328	Dissolve in PBS to prepare 0.1% Tween 20/PBS.