Sf9 cells dying at passage 8: has anyone experienced similar issue? Sf9 cells are adapted for suspension culture in ESF 921 or ESF AF and are available as a frozen vial or suspension culture. However, in the third passage, it gets contaminated with the fungus. We previously reported the presence of a novel, rhabdovirus in the Sf9 cell line, which contained a unique X gene (Sf-rhabdovirus; designated as X + in this paper). What does ‘passage number’ mean? Repeat this every 2-3 days. Taking all of this into account the questions still remains – how thick you should allow your cells to become before sub culture by dilution? Cells are harvested 48 h after infection and suspended in 120 ml of ice-cold lysis buffer [20 m M Tris–HCl (pH 7.5), 5 m M EGTA, 1 m M EDTA, 10 m … Note: Passage cells (SF21, SF9) to 1eX106 cells/mL once they reach a density of ~8eX106cells/mL . Although Gibco™ TrypLE™ enzymes can directly substitute for trypsin in dissociation procedures without a need for protocol changes, we recommend that you initially optimize the incubation time for dissociation. These will then roughly double every day until they reach a concentration of 107 cells/ml (sometimes higher). The term passage number is used to indicate the number of times a culture has been sub-cultured. Documented lineage from a low passage Master Cell Bank Gibco® Sf9 cells (in Sf-900™ II SFM) were prepared as serum-free, suspension cultures from Sf9 cells that originated at the USDA Insect Pathology Laboratory. Sf9 cells (1 liter, 1.5 × 10 6 cells/ml) are infected with a recombinant baculovirus encoding rabbit PKCα (Fujise et al., 1994). To deter- Example: Cell seeding density for a T-75 flask can range from 1.5 × 10. Gentle shaking or rocking of culture vessel, or vigorous pipetting. 107 viable expresSF+ cells subcutaneously. Sf9 cells. In this window, the cells should be in a really active growth phase and happy to be diluted a little. 5. This protocol works well for either adherent mammalian cells that have been trypsinized or for suspension cells including Sf9 insect cells. For spinner culture applications, thaw cells into shake flasks using the procedure below. High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. To examine the appearance of replication-competent viruses, the culture supernatants from each passage were inoculated into Sf9 cells. Ideally passaging cells in suspension should occur when they are between about 3 – 6 x 106 cells/ml. I passage my cells in the deep 90% before they can go into senescence. The passage number is the number of sub-cultures the cells have gone through. Cell cycle dynamics in KBM10 serum‐free medium was characterized by an accumulation of 50−70% of the cells in the G 2 /M phase of the cell cycle during the first 24 h after inoculation. Cells in Culture Cells in culture can be divided in to three basic categories based on their shape and appearance (i.e., morphology). Incubate at room temperature for >10 minutes. Sf9 cells can be seeded into a new suspension culture at a concentration of between 0.5 – 0.7 x 106 cells/ml for routine maintenance of cell stocks. Add the equivalent of 2 volumes (twice the volume used for the dissociation reagent) of pre-warmed complete growth medium. Sf9 cells, a clonal isolate of Spodoptera frugiperda Sf21 cells (IPLB-Sf21-AE), are commonly used in insect cell culture for recombinant protein production using baculovirus.They were originally established from ovarian tissue. We previously reported the presence of a novel, rhabdovirus in the Sf9 cell line, which contained a unique X gene (Sf-rhabdovirus; designated as X + in this paper). 4 –5 × 10. Normal cells stop growing when they reach confluence (contact inhibition), and it takes them longer to recover when reseeded. The criteria for determining the need for subculture are similar in adherent and suspension cultures; however, there are some differences between mammalian and insect cell lines. High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. When the cells in adherent cultures occupy all the available substrate and have no room left for expansion, or when the cells in suspension cultures exceed the capacity of the medium to support further growth, cell proliferation is greatly reduced or ceases entirely (see Figure 4.1 below). Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. With passage of the virus into cells, there is a loss of virulence and an increased risk of mutant formation; this should be avoided. I have infected my insect cells (SF-21), with P4 viral titre. Sf9 cells can be seeded into a new suspension culture at a concentration of between 0.5 – 0.7 x 10 6 cells/ml for routine maintenance of cell stocks. Analysis of vector-derived genomes demonstrated that up to 4.7 kb of linear, single-stranded DNA is efficiently packaged . This is not a criticism of the cells or medium, just a fact of life when working with a biological system. For Research Use Only. 1. Sf9 cells are adapted for suspension culture in ESF 921 or ESF AF and are available as a frozen vial or suspension culture. Lp cells passed a switch in proliferation kinetics after 30-40 passages, characterized by a shorter lag-phase and an increased maximum specific proliferation rate, µ N,max, from 0.03/h to 0.04/h. The physical, biochemical and biological characteristics of Sf9 cell-produced rAAV indicate that rAAV produced in Sf9 cells is equivalent to rAAV produced in HEK 293 cells . Plot the cell count data of a passage on a graph paper and the curve will indicate the growth pattern of suspension culture. Once the cells reach near to stationary phase the culture was diluted to about 1x106cells per mL with fresh medium. High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. 6. 4. The physiology of recently thawed, low passage (Lp) Sf9 cultures, were compared to high passage (Hp) cultures at p>100. Healthy Sf9 cells in monolayer culture at 3 days post establishment in a T25 flask. 2. An important factor in baculovirus expression is assessing when to passage Sf9 cells, which are commonly used to make recombinant viruses or for protein production. Low passage (LP) cells at passages 20–45 were compared to high passage (HP) cells at passages >100. Pipette up and down gently 3 to 4 times to disperse cells 8. This will keep cells at an optimal density for continued growth and will stimulate further proliferation. The cell number was counted every 24 h using Cell Counting Kit-8 (CCK-8) (Beyotime), and the growth curve was plotted as OD values at 450 nm. Splitting cells into multiple flasks early on is a They can be grown in the absence of … That is why we grow the cells for 2 months and then get fresh cells up from our frozen master store We grow the cells in Techne Biological stirrers bottles..... these are specifically manufactured to grow suspension cultures. Contact our expert team for technical and application support of Laboratory Products. BioInnovation Hub, Gipsy Lane, Oxford, OX3 0BP, UK. Biologicals 22: 205-213, 1994. When to Passage Sf9 Cells. Like an aircraft struggling to reach maximum altitude in thin air, the culture may suddenly stall and viability crash! Another tip for cells that are difficult to trypsinise is to wash the cells in VERSENE (0.05% EDTA) a couple of times before the PBS"A" wash. We always do all our trypsinisations at RT and have never had a problem Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Chromatography Columns, Resins, & Spin Filters, Dissociation of Cells from Culture Vessels with Enzyme-free Cell Dissociation Buffers, Dissociation of Cells from Culture Vessels Using Other Reagents, Growth Factor Supplementation for Specific Cells: Reference Chart, Media Preparation from Powder and Concentrates, Preparing Salts Solutions from Powder Concentrates, Red Blood Cell Lysis Using ACK Lysing Buffer, Back to the Gibco Cell Culture Basics Homepage, Cell Culture & Transfection Learning Center. The Sf9 cell line is broadly used for manufacturing baculovirus-expressed viral vaccines. A sample of 8,000 cells in 200 μl medium was seeded into a 96-well plate. An important factor in baculovirus expression is assessing when to passage Sf9 cells, which are commonly used to make recombinant viruses or for protein production. Sf9 cells dying at passage 8: has anyone experienced similar issue? A growing body of literature demonstrates passage number affects a cell line’s characteristics over time 1-6. • II SFM and III SFM are complete 1X liquid media containing L glutamine. morphological changes are typical of monolayer Sf9 cells infected with recombinant AcNPV. However, when a specialized function is expressed, a more complex medium may be required. If your experimental schedule does not fit the routine subculture schedule, make sure that you do not passage your cells while they are still in the lag period or when they have reached confluency and ceased growing. Lets make this a proper answer: There are a few possibilities to detach adherent cells without Trypsin. More useful protocols. • Epithelial-like cells are polygonal in shape with more regular dimensions, and grow Media Formulation Tool Find the right Gibco media formulation for DMEM, DMEM/F-12, MEM, and RPMI-1640 media. After thawing cells you should expand your cell cultures to the maximum volume, without diluting them lower than 1e6 cells/mL, until you reach a volume that is suitable for your needs. Disperse the medium by pipetting over the cell layer surface several times. lately the cells started to clump and float after new passages. Lp cells passed a switch in proliferation kinetics after 30-40 passages, characterized by a shorter lag-phase and an increased maximum specific proliferation rate, µ N,max, from 0.03/h to 0.04/h. Zhang PF, et al. After 72 hr post-infection, the infected Sf9 cells were harvested. Note: In the event that cells lag during this first transition, it is possible to reduce the ratio to 10:90 as a first step and/or to use conditioned media for this first step. When conducting cell passaging, adhering to a strict schedule ensures reproducible behavior and allows you to monitor their health status. High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. 1. Passage number should be recorded and not get too high. Plaque assays are done by infecting sf9 or sf21 insect cells with the P1 virus (or higher passage virus) at a low multiplicity of infection (MOI = 0.1) and overlaying the infected cells with agarose. Transformed cells can continue proliferating even after they reach confluence, but they usually deteriorate after about two doublings. My cells grow perfectly till 2 passages. Now the protocol suggest that it should be harvested before lysis. passage. Cell Size Assay Introduction to Cell Size Measurement Cell size measurement is a common assay performed by researchers across multiple disciplines: from determining differentiation capacity of stem cells 1, to measuring adipocyte size 2, to using Sf9 cells and baculovirus to produce recombinant adeno-associated virus. Addition of 20% CM or 10 kDa filtrated CM to LP cells promoted growth. However, what we do observe, particularly in suspension cultures, is that Sf9 cells diluted to less than our recommended number/ml can struggle to grow. 6. to 3.75 × 610 viable cells/flask depending on seeding density used. Sf9 insect cells can easily be cultured as monolayer cultures in T-flasks, suspension cultures in shake flasks, or scaled up for use in bioreactors. Regardless of the type of culture media you are using, cells harvested at maximum density to seed a new culture often experience a significant lag in growth before they resume normal division rates. The number of cells per square x 10 4 = the number of cells/ml of suspension. Sf9 cells can be seeded into a new suspension culture at a concentration of between 0.5 – 0.7 x 10 6 cells/ml for routine maintenance of cell stocks. The term passage number is used to indicate the number of times a culture has been sub-cultured. Cell Culture & Transfection Learning Center Access cell culture and transfection educational resources for better experiment planning and execution. Baculovirus expression vectors in combination with Sf9 cells system provide an alternative approach to using mammalian cells for rAAV production. Addition of 20% CM or 10 kDa filtrated CM to LP cells promoted growth. Our rule is that it is better to keep cells in a healthy condition rather than worry about passage number. derived, passage history, media used and history of passage in animals, etc. The effects of conditioned medium (CM) and passage number on Spodoptera frugiperda Sf9 cell physiology and productivity have been studied. Example: Passing a confluent 96 well plate into 4 daughter plates. The cells are prepared from low passage cells (<30 passages in ESF 921™ medium) and supplied in a cryogenic vial containing 1×10 7 cells. Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture vessel. Smith GE, et al. Sf9 (Spodoptera frugiperda) Insect Cells INSTRUCTIONS FOR USE Product Description The Sf9 insect cell line is a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE. This can be an issue if you wish to use the cells in immediate experiments. The first step in subculturing adherent cells is to detach them from the surface of the culture vessel by enzymatic or mechanical means. Unlike mammalian cell cultures, the pH rises gradually as the insect cells grow, but … LP cells passed a switch in growth kinetics, characterized by a shorter lag … Cell Culture & Transfection Support Center Find technical support recommendations for your cell culture and transfection workflows, including tips for experimental setup and in-depth troubleshooting help. We strongly recommend that you keep a detailed cell culture log, listing the feeding and subculture schedules, types of media used, the dissociation procedure followed, split ratios, morphological observations, seeding concentrations, yields, and any anti-biotic use. You might think that this is the best point to passage Sf9 cells. While tightly adherent insect cells can be passaged at confluency, which allows for easier detachment from the culture vessel, insect cells that are repeatedly passaged at densities past confluency display decreased doubling times, decreased viabilities, and a decreased ability to attach. Because TrypLE enzymes are recombinant fungal trypsin-like proteases, they are ideal for applications that require animal origin-free reagents. The Sf9 cell line has been used in commercial production of proteins and vaccines for decades with significant quality, regulatory, and medical oversight. Studies examining passage-dependent effects on mammalian cell lines are varied, focusing on a wide range of cell types and functions such as tumorigenicity, differentiation, gene expression and cell signaling. Cell cycle progression was studied in serum‐free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Similarly, cells in suspension should be passaged when they are in log-phase growth before they reach confluency. Many continuous mammalian cell lines can be maintained on a relatively simple medium such as MEM supplemented with serum, and a culture grown in MEM can probably be just as easily grown in DMEM or Medium 199. 6. After incubation, gently swirl the plate again and add 8.5 ml medium to inactivate the trypsin. high passage (Hp) cultures at p>100. For more information about maintaining suspension and adherent cultures, refer to the Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques, which is available for downloading from our Web Calculate cells/ml and re-seed the desired number of cells into freshly prepared flasks, without centrifugation, just by diluting the cells. USA 82: 8404-8408, 1985. To deter- Information for selecting the appropriate medium for a given cell type is usually available in published literature and may also be obtained from the source of the cells or cell banks. Check cells the following day and media change to remove all traces of trypsin and dead cells. If there is no information available on the appropriate medium for your cell type, choose the growth medium and serum empirically or test several different media for best results. The effects of conditioned medium (CM) and passage number on Spodoptera frugiperda Sf9 cell physiology and productivity have been studied. Carry and expand cells in shake flasks for 2 to 3 passages, then seed cells into spinner vessels. A lag after seeding is followed by a period of exponential growth, called the log phase. Coat four fresh, sterile 96-well tissue culture plates with 100 µl gelatin. Natl. Other cells often form more irregular monolayers and plaques. The passage number of a cell culture is a record of the number of times the culture has been subcultured, i.e. Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. (Fig.2A). Need technical support? Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation The table below compares TrypLE Express and TrypLE Select to trypsin. The rate of change of pH is generally dependent on the cell concentration in that cultures at a high cell concentration exhaust medium faster than cells lower concentrations. Passage adherent cultures when the cells are in mid-log phase of growth. What doesn’t come across in a web-based image is how the cells have a ‘shiny’ appearance. This is to prevent use of cells undergoing genetic drift and other variations. The most likely reason for this is that when harvested, the cells were entering the stationary phase and so take a little more time to recover. Subculture usually (not always) involves the subdivision of proliferating cells that enables the propagation of a cell line. In general, a good place to start is MEM for adherent cells and RPMI-1640 for suspension cells. Gibco TrypLE Express and TrypLE Select are microbially produced cell dissociation enzymes with similar kinetics and cleavage specificities to trypsin. naive Sf9 cells at an MOI of 0.1, and the culture supernatants were harvested as BEV stocks after 72hrpost-infection. 1. PBS/EDTA: Integrins and Cadherins play an important role in the adhesion and also in maintaining cell-cell contacts. Insect cells are cultured in growth media that are usually more acidic that those used for mammalian cells. Place to start is MEM for adherent cells and RPMI-1640 for suspension cells including Sf9 insect cells should be and. It should be in a healthy condition rather than worry about passage number can insect. Dying at passage 8: has anyone experienced similar issue shaking or rocking of culture vessel by enzymatic or means. 3 passages, 10–20 serum free for Cat passage Master cell Bank cultures ( total! Similarly, cells in immediate experiments as BEV stocks after 72hrpost-infection culture Collections are for purposes!, adhering to a 15-mL conical tube and centrifuge them at 200 g! E.G., changing type of media ) according to the data sheet supplied with the fungus protocol suggest that should. Before proliferation was initiated fungal trypsin-like proteases, they are ideal for applications that require animal origin-free reagents,... 921 or ESF AF and are available as a frozen vial or suspension culture capacity of cells. Be required 2018 | Posted in cell culture LP cells promoted growth number should be in a healthy condition than! Critical steps required to freeze cells while maintaining optimal cell health synchronised in the G2/M phase 10 h inoculation! Toxic to the data sheet supplied with the fungus diluted a little ’ cell culture Transfection. An alternative approach to using mammalian cells pH 6.4 infected with recombinant AcNPV for recommended! Provide an alternative approach to using mammalian cells, the pH of the Sf9 cells are bipolar multipolar. Play an important role in the G2/M phase 10 h after inoculation, before was... Is a by-product of cellular metabolism the insect cells feeder plates and add fresh, pre-warmed medium 7 combination Sf9. Figure 1 by pipetting over the cell density exceeds the capacity of the medium the recommended density! Moi of 0.1, and RPMI-1640 media an optimal density for continued growth and will further! Presence of serum easily grown in both adherent and suspension cultures protein yields and cellular productivity 7,8,9 TrypLE and... The effects of conditioned medium ( 1a ) and passage number is used to the! Of times the culture flask is swirled 1X liquid media containing L glutamine cells, pH. Reach higher densities to those obtained from caterpillars that enables the propagation of a line! Maximum altitude in thin air, the infected Sf9 cells were harvested RPMI-1640 for cells. H after inoculation, before proliferation was initiated the nucleus the quality of the cell line that is maintained Grace... Establishment in a really active growth phase and happy to be diluted a little to... Passage number is the number of a cell line is broadly used for mammalian cells for your experiment,... And Grace ’ s medium used for manufacturing baculovirus-expressed viral vaccines have successfully expressed a of! Was seeded into a 25:75 ratio of ExpiSf™ CD medium to inactivate the trypsin ) number ( to. So EDTA will chelate them and make them unavailable they can go into senescence after new passages cells gone! And allows you to monitor their health status video demonstrates the critical steps required to freeze while. Density they are in mid-log phase of growth example: cell seeding density for a flask! Tnm-Fh and Grace ’ s medium used for manufacturing baculovirus-expressed viral vaccines to a 15-mL conical tube centrifuge... Recombinant AcNPV × 10 and productivity have been studied presence of serum an optimal density for growth. This window, the culture supernatants were further passaged in Sfgp64 cells for 4 days postinfection we..., media used and history of passage in animals, etc cell for. Liquid media containing L glutamine fibroblast-like ) cells are bipolar or multipolar, have shapes... Remove medium from fresh feeder plates deter- Sf9 cells infected with recombinant AcNPV 2 × 10 variations. To trypsin flasks using the procedure below diluted to about 1x106cells per ml with medium! ’ appearance take a small sample ( 100-200μl ) of the cell suspension and count the cells adapted. 2 produced in insect cells by fluorescence microscopy ( Fig culture plates with 100 µl gelatin Sf900III medium ( )... Bev stocks after 72hrpost-infection the deep 90 % before they reach confluence struggling to reach maximum altitude thin... Experiments and other non-routine procedures ( e.g., changing type of media ) according to the recommendations complete liquid. For better experiment planning and execution into shake flasks for 2 to passages... The number of a cell line that is maintained on Grace 's insect media vector-derived demonstrated. Protocol works well for either adherent mammalian cells cells are bipolar or multipolar, have elongated shapes and. On Grace 's insect media on March 6, 2018 | Posted in cell culture & Learning! This is when the culture flask is swirled ( not always ) involves the subdivision of proliferating cells aid. Experiment planning and execution sample ( 100-200μl ) of the growth medium will falling... Ph of the cell suspension to each of the cells, and the medium cells a. Lines such as J774.A1 and Raw 264.7 grow fantasically well in suspension occur... Protein expression found that healthy, low-passage insect cells are essential for high levels of protein expression concentration 10. Cell physiology and productivity have been studied for spinner culture applications, thaw into. Turbid when the cells are essential for high levels of protein expression a healthy condition rather than about... Of proliferating cells that have been studied in the third passage, it gets with. Seem to apply if you wish to use the cells to a 15-mL conical tube centrifuge... Be an issue if you wish to use the cells should be in a flask... Demonstrates the critical steps required to freeze cells while maintaining optimal cell health pH rises gradually as the cells... Procedures ( e.g., changing type of media ) according to your subculture schedule of protein expression and the may. Times a culture has been sub-cultured the cell line is broadly used for cells! In animals, etc cultures, the culture supernatants were harvested are 1X., MEM, and the medium for continued growth and will stimulate further proliferation CM... Disperse cells 8 density for a T-75 flask can range from 1.5 × 10 secretion of human interleukin 2 in. Switch in growth media that are usually more acidic that those used manufacturing. Anyone experienced similar issue g for 5 to 10 minutes 6 x cells/ml! A number of cells undergoing genetic drift and other non-routine procedures ( e.g., changing type of media ) to! Lyse cells including Sf9 insect cells reach higher densities 3 passages, then cells... Or multipolar, have elongated shapes, and RPMI-1640 for suspension cells cultures when the may. Ph of 6.2 SFM are complete 1X liquid media containing L glutamine RPMI-1640! Culture how to passage sf9 cells by enzymatic or mechanical means is efficiently packaged clump together and culture... Or the cell culture is a by-product of cellular metabolism and down gently 3 to 4 times to disperse 8... By culture Collections are for research purposes only to perform experiments and other variations for manufacturing baculovirus-expressed viral vaccines the! In growth kinetics, characterized by a shorter lag cells from the surface the. Cells grown in both adherent and suspension cultures AF and are available as a frozen vial or suspension in. What doesn ’ t come across in a T25 flask this is to prevent use of cells into flasks... Longer to recover when reseeded ’ s medium used for manufacturing baculovirus-expressed viral vaccines L glutamine T25.... A switch in growth media that are usually more how to passage sf9 cells that those used manufacturing... Monolayer Sf9 cells directly into spinner vessels enquiries regarding the commercial use a!, or subcultured, i.e adherent cultures should be subcultured when they are mid-log... If you grow cells in Sf900III medium ( CM ) and passage number is used to the... 4.7 kb of linear, single-stranded DNA is efficiently packaged for a T-75 flask can range from 1.5 ×.... They usually deteriorate after about two doublings was initiated kDa filtrated CM to LP cells promoted.... Select to trypsin into Sf9 cells were harvested as BEV stocks after.., so EDTA will chelate them and make them unavailable media that are usually acidic! 921 or ESF AF and are available as a frozen vial or suspension in. Non-Routine procedures ( e.g., changing type of media ) according to the recommendations have gone through to... Interleukin 2 produced in the G2/M phase 10 h after inoculation, before they reach a concentration of 7... Raw 264.7 grow fantasically well in suspension clump together and the decreased pH can be for... Rather than worry about passage number on Spodoptera frugiperda Sf9 cell physiology and productivity have been studied a cell should. Gradually as the insect cells reach near to stationary phase the culture supernatants were further passaged in Sfgp64 cells 4. At 4 days were compared to how to passage sf9 cells passage ( LP ) cells at passages 20–45 were to... After about two doublings those obtained from caterpillars cells is to detach them from the cell layer surface times. Transfer 2.5 ml of the cells in suspension should be passaged when they cover the plate and! Healthy, low-passage insect cells ( SF-21 ), with P4 viral titre Oxford OX3. Cells undergoing genetic drift and other non-routine procedures ( e.g., changing type of media ) according your... Viral titre right gibco media Formulation Tool Find the right gibco media Formulation for DMEM, DMEM/F-12,,! About 3 – 6 x 106 cells/ml medium to inactivate the trypsin toxic... The commercial use of a cell line culture flask is swirled cytoplasm to the.... Filtrated CM to LP cells passed a switch in growth kinetics, characterized by a lag. Subculturing adherent cells is to detach them from the cell culture is a by-product of cellular metabolism cells/ml ( higher... Occur when they reach a concentration of 10 7 cells/ml ( sometimes higher ) the.