Reference

Cell Culture Reference Library

A working reference for the field. What each medium, buffer, reagent and technique actually is, what is in it, and how it differs from the alternatives — written for the bench, not for a brochure.

Media and formulations

Chemically Defined Medium

A chemically defined medium is a cell culture medium in which the identity and concentration of every component is known — no serum, no protein hydrolysates, no undefined extracts. It may still contain proteins, provided they are recombinant and of known sequence and concentration, which is why chemically defined and protein-free are distinct categories rather than synonyms. Chemically defined media eliminate the lot-to-lot variability, adventitious agent risk and regulatory burden of serum, and are the standard for biopharmaceutical production in CHO, HEK293 and hybridoma systems.

RPMI 1640 Medium

RPMI 1640 is a basal cell culture medium developed at Roswell Park Memorial Institute in 1966 for the culture of human leukocytes in suspension. It contains 2,000 mg/L glucose (11.1 mM), 2,000 mg/L sodium bicarbonate (23.8 mM) buffered for a 5% CO2 atmosphere, unusually high phosphate (about 5.6 mM), low calcium (about 0.42 mM), and a distinctive component set that includes reduced glutathione, biotin, vitamin B12, para-aminobenzoic acid and hydroxyproline. It is the standard medium for lymphocytes, hybridomas, and most suspension-adapted haematopoietic and lymphoid cell lines, normally supplemented with 10% fetal bovine serum.

MEM Alpha (α-MEM) and Minimum Essential Medium

MEM alpha (α-MEM) is Eagle's Minimum Essential Medium enriched with all the non-essential amino acids, sodium pyruvate, lipoic acid, ascorbic acid, biotin and vitamin B12, and is supplied either with or without ribonucleosides and deoxyribonucleosides. It uses the same salt base as MEM — 2,200 mg/L sodium bicarbonate matched to a 5% CO2 atmosphere, 1,000 mg/L glucose and 1.8 mM calcium — and is the standard medium for mesenchymal stromal cells, bone marrow cultures, osteoblasts, CHO-DXB11 and CHO-DG44 selection, and many primary cell types. The nucleoside-free version is required for HAT and methotrexate-based selection systems.

DMEM (Dulbecco's Modified Eagle Medium)

DMEM (Dulbecco's Modified Eagle Medium) is a basal cell culture medium derived from Eagle's Minimal Essential Medium by raising the amino acid and vitamin concentrations roughly fourfold. It is supplied in high-glucose (4,500 mg/L, 25 mM) and low-glucose (1,000 mg/L, 5.6 mM) forms, buffered with 3,700 mg/L sodium bicarbonate, and requires serum or a defined supplement plus a CO2 atmosphere to hold physiological pH. DMEM is the default medium for adherent lines such as HEK293, HeLa, NIH/3T3, Vero and CHO-derived adherent cultures, and for most primary fibroblasts.

Phenol Red in Cell Culture Media

Phenol red (phenolsulfonphthalein) is a pH indicator added to cell culture media at roughly 5-15 mg/L, where it turns yellow below about pH 6.8 and pink to fuchsia above about pH 8.2, with the normal red-orange of healthy medium sitting near pH 7.4. Medium turning yellow means acidification - almost always lactate from cell metabolism, from an overgrown culture or from bacterial contamination - while medium turning pink or purple means the medium has gone alkaline, nearly always because CO2 has escaped from the bicarbonate buffer. Phenol red does no buffering itself and is purely diagnostic; use phenol-red-free medium for fluorescence and absorbance assays, and for oestrogen-responsive cell work, where the dye interferes with the readout.

Reagents and buffers

HEPES buffer in cell culture

HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a zwitterionic organic buffer used in cell culture to hold medium at physiological pH without depending on incubator CO2. Its pKa is about 7.5 at 20-25 degrees C and about 7.3 at 37 degrees C, giving useful buffering across roughly pH 6.8-8.2, and it is normally added to medium at 10-25 mM from a sterile 1 M stock. Because HEPES buffering does not rely on the carbonic acid/bicarbonate equilibrium, HEPES-supplemented medium resists the fast alkaline drift that occurs when a flask leaves a 5% CO2 incubator.

HBSS (Hank's Balanced Salt Solution)

HBSS (Hank's Balanced Salt Solution) is an isotonic balanced salt solution used to wash cells, transport tissue, dilute reagents and hold cells briefly outside their growth medium. It contains 8.0 g/L sodium chloride, 1.0 g/L D-glucose, phosphate and 350 mg/L sodium bicarbonate, and is supplied either with calcium and magnesium (1.26 mM Ca, ~0.9 mM Mg total) or without them. Its low bicarbonate means it is designed for use at atmospheric CO2 or in sealed vessels, not for prolonged culture in a 5% CO2 incubator.

Trypan Blue and the Dye Exclusion Viability Assay

Trypan blue is a diazo dye used at 0.4% (w/v) to separate live from dead cells by dye exclusion: an intact plasma membrane keeps the charged dye out, so viable cells stay clear and refractile, while cells with a damaged membrane take the dye up and stain blue. In practice the cell suspension is mixed 1:1 with 0.4% trypan blue, loaded into a hemocytometer or an automated cell counter, and counted within 3-5 minutes to give both a cell concentration and a percent viability. Because it reports membrane integrity only, trypan blue counts cells that have already died -- it does not detect apoptotic or metabolically failing cells that still have an intact membrane, so it reads high compared with metabolic or flow-cytometry viability assays.

ACK lysis buffer

ACK (Ammonium-Chloride-Potassium) lysis buffer is an isotonic ammonium chloride solution used to remove red blood cells from leukocyte preparations such as mouse spleen, bone marrow and buffy coat. The standard composition is 150 mM ammonium chloride (8.02 g/L NH4Cl), 10 mM potassium bicarbonate (1.0 g/L KHCO3) and 0.1 mM disodium EDTA, adjusted to pH 7.2-7.4. Red cells lyse osmotically within one to five minutes at room temperature because their band 3 anion exchanger and high carbonic anhydrase activity drive ammonium chloride and water into the cell, while leukocytes, which lack that transport capacity, survive.

Trypsin-EDTA

Trypsin-EDTA is a cell dissociation reagent that combines the serine protease trypsin, which cleaves peptide bonds on the C-terminal side of lysine and arginine residues in cell-surface and matrix proteins, with the chelator EDTA, which binds the calcium and magnesium ions that cell-adhesion molecules require. It is supplied in a calcium- and magnesium-free balanced salt solution, most commonly at 0.05% trypsin (0.5 g/L) for routine cell lines and 0.25% (2.5 g/L) for firmly adherent cells and primary cultures. Typical use is 2-5 minutes at 37 degrees C, followed immediately by neutralisation with serum-containing medium or a defined trypsin inhibitor.

Puromycin for Mammalian Cell Selection

Puromycin is an aminonucleoside antibiotic from Streptomyces alboniger that mimics the aminoacyl end of tRNA, enters the ribosomal A site and causes premature release of a truncated peptide chain, killing cells that cannot inactivate it. In mammalian cell culture it is used as a selection agent at 0.5-10 ug/mL, most commonly 1-2 ug/mL, and it kills non-resistant cells faster than any other common selection antibiotic -- typically within 2-5 days. Resistance is conferred by the pac gene encoding puromycin N-acetyltransferase, which acetylates the drug and renders it inactive. Because the effective concentration depends strongly on cell line, cell density and serum content, a kill curve must be run for each new cell line and each new lot of puromycin.

Penicillin-Streptomycin and Antibiotic-Antimycotic in Cell Culture

Penicillin-streptomycin, universally shortened to pen-strep, is supplied as a 100X sterile solution containing 10,000 units/mL penicillin G and 10,000 ug/mL streptomycin, diluted 1:100 into medium to give a working concentration of 100 U/mL penicillin and 100 ug/mL streptomycin. Penicillin blocks bacterial cell wall synthesis and covers mainly Gram-positive organisms; streptomycin binds the bacterial 30S ribosomal subunit and covers mainly Gram-negatives, so the pair gives broad antibacterial coverage. Antibiotic-antimycotic (anti-anti) is the same combination plus 25 ug/mL amphotericin B at 100X, giving 0.25 ug/mL in use for antifungal coverage. Neither controls mycoplasma, and major cell banks including ATCC recommend against routine antibiotic use because it masks low-level contamination rather than preventing it.

Bovine Serum in Cell Culture

Bovine serum is the liquid fraction of clotted cattle blood, added to culture medium at 5-10% to supply growth factors, hormones, transport and attachment proteins, lipids and trace elements that basal media do not contain. Fetal bovine serum (FBS) and fetal calf serum (FCS) are two names for the same product, collected from the fetus at slaughter of pregnant cows; newborn calf serum comes from calves under about 20 days old and donor bovine serum from controlled donor herds aged roughly 12-36 months, both containing more immunoglobulin and fewer growth factors than FBS. Because serum is an undefined biological material with substantial lot-to-lot variation, unresolved animal welfare questions and a volatile supply chain, defined serum-free and animal-origin-free media are increasingly preferred where the cell line will tolerate them.

Iodixanol as a Density Gradient Medium

Iodixanol is a non-ionic, water-soluble iodinated compound used as a density gradient medium, supplied as a sterile 60% (w/v) solution with a density of 1.32 g/mL. Its defining advantage is that it is iso-osmotic across the whole useful density range -- a 60% solution has an osmolality of approximately 290 mOsm/kg -- so cells, organelles, viruses and extracellular vesicles can be banded at high density without the osmotic dehydration caused by sucrose or the high ionic strength of caesium chloride. It is the standard medium for laboratory-scale AAV purification, where a four-step gradient of 15%, 25%, 40% and 60% separates genome-containing capsids at the 40-60% interface, and it is also widely used for cell separation, organelle fractionation and extracellular vesicle flotation.

Glucose solution in cell culture

A cell culture glucose solution is a concentrated sterile D-glucose stock, commonly supplied at 300-450 g/L (30-45% w/v), used to supplement basal media and to feed cultures that consume glucose faster than the medium supplies it. D-glucose has a molecular weight of 180.16, so 1 g/L equals 5.55 mM: standard media run from 1 g/L (5.5 mM) in low-glucose DMEM through 2 g/L (11.1 mM) in RPMI 1640 to 4.5 g/L (25 mM) in high-glucose DMEM. Glucose is added to prevent depletion in long or high-density cultures, and it is normally sterile-filtered rather than autoclaved, because heating glucose with amino acids produces browning reaction products.

Cell culture buffers

Cell culture buffers hold medium in the pH 7.2-7.4 range that mammalian cells require, against the acid load cells generate as they metabolise. The default system in almost every classical medium is sodium bicarbonate working with the CO2 in the incubator atmosphere, which is why bicarbonate content must be matched to the incubator setting: about 1.5-2.2 g/L NaHCO3 for 5% CO2 and 3.7 g/L for 10% CO2. Organic buffers such as HEPES (pKa 7.48) are added at 10-25 mM to hold pH when cultures are outside a CO2 atmosphere, while phosphate-buffered solutions such as PBS and DPBS are used for washing and short-term handling rather than for growth.

Recombinant Human Albumin in Cell Culture

Recombinant human serum albumin (rHSA) is a 585-amino-acid, 66.5 kDa non-glycosylated protein produced in yeast such as *Pichia pastoris* or in transgenic rice rather than purified from human plasma, and it is used as an animal-origin-free supplement in serum-free and chemically defined culture media. In culture it works as a carrier for fatty acids, lipids, hormones and trace elements, as an antioxidant through its free cysteine-34 thiol, as a scavenger that sequesters toxic metals and excess free fatty acids, and as a shear-protective surface-active protein in stirred and sparged culture. Typical working concentrations are 0.1-10 g/L, most often 0.5-5 g/L, and it is commonly supplied as a 200 mg/mL (20%) solution. Its performance depends heavily on what is bound to it, so fatty-acid-loaded and fatty-acid-free preparations behave quite differently.

Dispase

Dispase, also sold as Dispase II or neutral protease, is a zinc-dependent metalloprotease from Paenibacillus (formerly Bacillus) polymyxa, EC 3.4.24.4, used for gentle tissue dissociation and for detaching cells and epithelial sheets intact. It cleaves fibronectin and type IV collagen, degrades type I collagen only minimally, and does not cleave laminin or type V collagen, which is why it can separate an epidermis from a dermis or lift a confluent epithelial sheet off plastic without destroying cell-cell junctions. Because it is a metalloprotease requiring zinc for catalysis and calcium for stability, it is inhibited by EDTA, EGTA and 1,10-phenanthroline, and it must be diluted in a calcium-containing buffer rather than a chelator-based one.

Recombinant Human Insulin in Cell Culture

Recombinant human insulin is a 51-amino-acid, 5.8 kDa two-chain protein used as a core supplement in serum-free and chemically defined culture media, where it drives glucose and amino acid uptake, protein and lipid synthesis, and cell survival. It is produced in *E. coli* or yeast rather than extracted from animal pancreas, making it animal-origin-free, and is usually supplied as a zinc-stabilised dry powder. The typical working concentration is 5-10 ug/mL, the level delivered by a standard 1X ITS supplement, which is roughly a thousandfold above physiological insulin levels -- at that concentration insulin acts substantially through the IGF-1 receptor as well as its own. Because insulin is poorly soluble near neutral pH, powder must be dissolved in dilute hydrochloric or acetic acid before dilution into medium.

Sera, supplements and cell lines

Sf9 Cells and the Baculovirus Expression System

Sf9 is a clonal insect cell line isolated from Sf21, which was established from pupal ovarian tissue of the fall armyworm *Spodoptera frugiperda*. It is the standard host for the baculovirus expression vector system, used to produce recombinant proteins, virus-like particles and AAV. Sf9 cells grow at 27 C without CO2 in phosphate-buffered insect media at pH 6.2-6.4, adapt readily to serum-free suspension culture at 100-130 rpm, and double every 18-24 hours. Cultures are passaged when they reach roughly 2.5-3.5 x 10^6 cells/mL and diluted back to about 1 x 10^6 cells/mL, and infections are performed on healthy log-phase cells above 95% viability.

DF-1 Chicken Embryo Fibroblast Cells

DF-1 (UMNSAH/DF-1, ATCC CRL-12203) is a spontaneously immortalised chicken embryo fibroblast line derived from East Lansing Line 0 embryos, a chicken line free of endogenous avian leukosis virus loci. Because it carries no endogenous retrovirus and was immortalised without viral or chemical transformation, it is the standard continuous substitute for primary chicken embryo fibroblasts in avian virology, vaccine development and recombinant virus production. DF-1 cells are cultured in high-glucose DMEM with 10% fetal bovine serum at 37 C in 5% CO2, grow faster than primary CEF, transfect efficiently, and support replication of avian influenza, infectious bursal disease virus, avian leukosis and sarcoma viruses and many other avian pathogens at titres equal to or higher than CEF.

Protocols and methods

Counting Cells with a Hemocytometer

A hemocytometer is a thick glass slide bearing a precisely etched grid over which a coverslip creates a chamber exactly 0.1 mm deep, so that a known volume of cell suspension can be counted under a microscope and converted to a concentration. For mammalian cells you count the four 1 mm2 corner squares, take the mean, multiply by the dilution factor, and multiply by 10^4 to obtain cells per mL, because each corner square encloses exactly 10^-4 mL. Mixing the sample 1:1 with 0.4% trypan blue before loading gives a viability percentage from the same count, since only dead cells with compromised membranes take up the dye.

Heat Inactivation of Fetal Bovine Serum (FBS)

Heat inactivation of fetal bovine serum is a controlled 30-minute incubation at 56 degrees C that destroys the heat-labile proteins of the complement cascade before the serum is added to culture medium. The serum is thawed, brought to 37 degrees C, transferred to a 56 degrees C water bath, held for exactly 30 minutes from the moment the serum itself reaches temperature with gentle swirling every 5-10 minutes, then cooled immediately and aliquoted. For routine culture of established cell lines it is usually unnecessary, because complement activity in fetal serum is low and heating also degrades labile growth factors; it remains standard practice for immunological assays and complement-sensitive cells.

Subculture of Cells (Passaging)

Subculture of cells, also called passaging, is the transfer of cells from a culture that is approaching confluence into fresh vessels with fresh medium so that growth can continue. Adherent cells are detached first, usually with 0.25% or 0.05% trypsin-EDTA or a non-enzymatic dissociation reagent, then reseeded at a lower density set by a split ratio such as 1:4; suspension cells are simply diluted into fresh medium without any dissociation step. Most continuous adherent cell lines are subcultured at 70-80% confluence, which normally means two or three passages per week.

Freezing Medium for Cells and Cryopreservation Protocols

Cell freezing medium is a cryoprotectant-containing solution that allows cells to survive freezing and long-term storage, most commonly built from a base medium or serum plus 5-10% dimethyl sulfoxide (DMSO), with 10% the standard starting point. DMSO permeates the cell and modifies how water freezes, preventing the intracellular ice crystals that otherwise rupture membranes during cooling. Cells are frozen at a controlled rate of approximately 1 C per minute to -80 C and then transferred to the vapour phase of liquid nitrogen, since long-term storage requires temperatures below about -135 C to prevent ice recrystallisation. Thawing is the opposite -- as rapid as possible in a 37 C water bath, followed by prompt dilution to remove the DMSO, which is toxic to cells above freezing temperatures.

Poly-L-Lysine Coating for Cell Culture

Poly-L-lysine coating covers a culture surface with a positively charged synthetic polymer that binds the negatively charged plasma membrane, allowing weakly adherent cells to attach to glass or plastic. The standard protocol applies a sterile 0.01% (0.1 mg/mL) solution at roughly 1 mL per 25 cm2, leaves it in contact with the surface for 5 minutes to 1 hour at room temperature, aspirates, rinses thoroughly with sterile tissue-culture-grade water, and air-dries the surface for at least 2 hours before cells are seeded. The attachment it provides is purely electrostatic: poly-L-lysine does not engage integrins, so cells that need genuine extracellular matrix signalling require laminin, fibronectin or collagen instead of it or on top of it.

Comparisons

PBS vs DPBS: What Is Different, and Which One to Use

PBS and DPBS are both phosphate-buffered saline solutions; the difference is the recipe, not the function. Dulbecco's formulation (DPBS) adds potassium chloride and carries roughly twice the phosphate of a typical PBS (about 9.5 mM versus about 4 mM), and it is sold in two versions - with calcium and magnesium, and without. In practice the with-or-without-divalent-cations choice matters far more than the PBS-or-DPBS label: use a calcium- and magnesium-free solution to wash cells before trypsinisation or EDTA dissociation, because Ca2+ and Mg2+ support the cadherin and integrin bonds you are about to break, and use the version containing calcium and magnesium when cells must stay attached and intact through the wash.

Essential and Non-Essential Amino Acids in Cell Culture

In cell culture, an amino acid is called essential if cultured mammalian cells cannot make enough of it and it must be supplied in the medium - thirteen of them, the set Harry Eagle defined, including arginine, cystine, tyrosine and glutamine that are not classed as dietary essentials. The seven non-essential amino acids - glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine - can be synthesised by most cells, and are supplied anyway because making them consumes carbon, ATP and reducing equivalents that would otherwise go into growth. A MEM NEAA 100X supplement contains all seven at 10 mM, giving 0.1 mM of each at working strength; add it to MEM or DMEM, which contain few or none, and leave it out of Ham's F-12, IMDM and DMEM/F-12, which already carry all seven.

EMEM vs DMEM: What the Modification Actually Changed

EMEM (Eagle's Minimum Essential Medium, also sold as MEM) and DMEM (Dulbecco's Modified Eagle Medium) are the same medium one generation apart: DMEM is Eagle's formulation enriched, with roughly four times the vitamins, about twice most amino acids, twice the glutamine, added glycine and serine, ferric nitrate, and 3.7 g/L sodium bicarbonate against EMEM's 1.5-2.2 g/L. The practical consequence is that DMEM supports fast-growing, metabolically demanding lines such as HEK293 and NIH/3T3, while EMEM suits slower, less demanding adherent cells and primary lines - and because DMEM's higher bicarbonate is formulated for 10% CO2 while EMEM's suits 5%, the two are not interchangeable without checking your incubator.

Trypsin vs Accutase: Choosing a Cell Dissociation Reagent

Trypsin is a pancreatic serine protease that cleaves peptide bonds after lysine and arginine, and it detaches cells fast but indiscriminately - it must be stopped with serum or a trypsin inhibitor, and it cleaves surface proteins along with the adhesion contacts. Accutase is a proprietary mixture of proteolytic and collagenolytic enzymes from an invertebrate source, supplied in calcium- and magnesium-free DPBS with 0.5 mM EDTA; it works more slowly and gently, does not require a neutralisation step, and preserves more surface epitopes. Choose trypsin for routine passaging of robust adherent lines where speed matters and the cells go straight back into culture; choose Accutase when the harvested cells are the experiment - flow cytometry on surface markers, pluripotent stem cells, primary neurons, or any single-cell suspension where viability and phenotype must survive the harvest.

Contamination and quality control

Cell Culture Contamination

Cell culture contamination falls into six categories: bacteria, yeast, filamentous fungi and moulds, mycoplasma, viruses, and cross-contamination by another cell line. Bacteria, yeast and fungi announce themselves within one to five days through turbidity, a pH shift or visible particles under the microscope, and are handled by discarding the culture and finding the technique failure that let them in. Mycoplasma, viral contamination and cell line misidentification produce no visible change at all and are found only by testing — PCR or DNA stain for mycoplasma, STR profiling for identity. The correct response to any confirmed biological contamination is to discard the affected culture, decontaminate the incubator, and restart from a clean frozen stock.

Endotoxin Testing in Cell Culture

Endotoxin testing measures bacterial lipopolysaccharide (LPS) in laboratory water, media, sera and reagents, reported in endotoxin units per millilitre (EU/mL), where 1 EU corresponds to roughly 0.1–0.2 ng of reference-standard E. coli endotoxin. The compendial methods are the LAL (limulus amebocyte lysate) assay in its gel-clot, turbidimetric and chromogenic forms under USP <85>, and the animal-free recombinant Factor C (rFC) assay under USP <86>; kinetic formats quantify down to about 0.001–0.005 EU/mL. Practical cell-culture limits are 0.25 EU/mL for water, ≤1 EU/mL for most cell-culture-grade reagents, and ≤10 EU/mL as the general industry standard for fetal bovine serum, with low-endotoxin grades at ≤1 EU/mL and ultra-low grades below 0.1 EU/mL. Endotoxin survives autoclaving and passes 0.2 µm filters, so it must be excluded at source rather than removed later.

Mycoplasma Testing in Cell Culture

Mycoplasma testing detects Mollicutes species that contaminate laboratory cell cultures without producing turbidity, pH change or any other visible sign, which is why a culture can carry 10⁷–10⁸ organisms per mL and still look healthy. The practical methods are PCR or qPCR (roughly 1–10 CFU/mL, results the same day), enzymatic luminescence assays (about 20 minutes, moderate sensitivity), fluorescent DNA staining read against an indicator cell line (about 10²–10⁶ CFU/mL depending on whether an enrichment step is used), and direct broth-and-agar culture, which remains the reference method but takes up to 28 days. Test cultures on arrival, after quarantine and at least monthly thereafter; a confirmed positive is normally discarded and replaced from a clean frozen stock rather than treated.

Selection Antibiotics for Mammalian Cell Lines

Selection antibiotics are cytotoxic agents used to isolate mammalian cells that carry a co-transfected resistance gene, by killing every cell that does not. The five standard agents are G418/geneticin (100–2000 µg/mL, resistance gene neo), hygromycin B (50–1000 µg/mL, hph), blasticidin S (1–20 µg/mL, bsd or bsr), zeocin (50–1000 µg/mL, Sh ble) and puromycin (0.5–10 µg/mL, pac). The correct concentration is not the one on the datasheet but the one determined empirically for your cell line by a kill curve — the lowest concentration that kills 100% of untransfected cells within the agent's normal selection window, which is 2–4 days for puromycin and up to 14 days for G418.

Antibiotics and Antimycotics in Cell Culture

Antibiotics in cell culture are antibacterial and antifungal agents added to growth medium to suppress microbial contamination; the standard combinations are penicillin–streptomycin at 100 U/mL and 100 µg/mL, gentamicin at 5–50 µg/mL, amphotericin B as an antimycotic at 0.25–2.5 µg/mL, and antibiotic–antimycotic 100X, which supplies all three at 100 U/mL, 100 µg/mL and 0.25 µg/mL when diluted. The professional consensus is that these should not be used routinely or prophylactically: they mask low-level contamination, select for resistant organisms, do nothing at all against mycoplasma, and can alter cell physiology and experimental response. Their defensible uses are primary tissue isolation, work with material from non-sterile sites, and short-term rescue of an irreplaceable culture — not the standard maintenance of established cell lines.