60931_LM_Corning_Lab_Tips_custom_eBook_JD_V6 (1) Lab Life Essentials: Level Up with Top Tips for Everyday Tasks Increase productivity and repeatability and reduce costs in life science labs Introduction Efficiency and precision are cornerstones of success in today's fast- paced life science labs. This compendium of expert tips and tricks for everyday lab tasks is designed to equip you with knowledge and skills to enhance your productivity, improve repeatability, and streamline your experiments. These top tips cover a wide range of critical lab practices, from ensuring accuracy and reliability of pipetting-particularly with challenging liquids-to ensuring safe centrifugation and sample handling and optimal cell culture performance. Delving deeper into cell culture success, we will explore tips for optimizing cell growth, maintaining healthy cultures, preserving frozen cell viability, and preventing endotoxin contamination. Together, these practices safeguard your results, boost efficiency and quality, and even save money. Let's embark on a journey to become the ultimate laboratory MVP, acing daily tasks with style. . Table of Contents ❱ Top Pipetting Tips for Accuracy and Challenging Liquids 3 ❱ Top Safety Tips for Centrifuges and Centrifuge Tubes 7 ❱ Top Tips for Cell Culture 9 ❱ Top Tips for Maintaining Frozen Cell Viability 11 ❱ Top Tips to Prevent Endotoxin Contamination 13 Achieving reliable results when pipetting depends largely on the user's skills. Errors resulting from poor pipetting techniques can sig- nificantly affect test results, as proper pipetting plays an important role in achieving accurate and repeatable measurements. Proper pipetting Select the most appropriate pipettor Example: When you want to pipet a 10 µL sample, use a pipettor with the 0.5 to 10 µL volume range rather than a pipettor with the 10 to 100 µL volume range. The accuracy error is lower in the first case. Using pipettors at, or close to, the nomi- nal volume settings is particularly important when pipetting liquids at temperatures above or below room temperature. Pay attention to environmental conditions The volume of liquid measured using a pipettor varies with the temperature. The pipettor, the pipet tip, and the liquid should be constant in the range of 20°C to 25°C when pipetting at room temperature because the devices and consumables are calibrated at this temperature. Prevent cross-contamination using filter tips Filter tips are the best solution for applications demanding the highest level of purity (radioactive solutions, proteins, nucleic acids, cell cultures, etc.). They let you protect both the pipettor and the sample from cross-contamination. A filter prevents liquid from making contact with the pipettor's internal lower parts when aspirating, and also it prevents aerosols from alkalis or acids entering into the pipet shaft, which could damage the device. Change the tip Always replace the tip with a new one when changing dispensed liquid or if visible liquid droplets remained inside the tip. Applying the same tip repeatedly may result in an error of approximately four percent. Clean and calibrate the pipettor regularly Pipetting challenging liquids External surfaces of the pipettor may be cleaned using a cloth dampened in isopropyl alcohol. Calibrate your pipettors at least once each year to ensure that the parameters are consistent with the specifications. Use proper pipetting technique - Hold the pipettor vertically when aspirating the liquid. Positive-displacement System Piston Disposable syringe tip Sample Corning Step-R Repeating Pipettor Use a positive-displacement system for challenging liquids Most commonly used pipettors (e.g.,Corning®Lambda™Plus Pipettor) work based on an air-cushion principle and are perfect for most pipetting applications. Consider tip pre-wetting When your pipetting volumes are greater than 10 µL pre-wetting a pipet tip is highly recommended. Just aspirate some liquid and then dispense it back into the original container. Pre-wetting is recommended in most preparations for improved accuracy. Store the pipettor on a stand Leaving the pipettor flat on the bench, especially with liquid in the tip, could cause the liquid to enter the pipettor and result in corrosion of internal parts. Also important is the warmth of the hand during pipetting, as this can have an impact on the thermal equilibrium and consequently influence the volume of the dispensed sample. Therefore, do not hold the pipettor continuously in your hand between pipetting cycles. However, the precision and accuracy of the pipetting results can be affected when working with liquids of different temperatures or with a different viscosity, volatility, or density than water. In these cases, positive-displacement pipettors (e.g., Corning Step-R™ Repeating Pipet- tor) can improve the pipetting process and subsequent results because there is no air cushion and no variation of the volume aspirated in response to the physical properties of samples. Recalibrate air-displacement pipettors for differing liquid density Higher density liquids have greater mass per unit volume and impose an increased gravitational force on the air space between the liquid and the piston. The increased air space results in a smaller volume of liquid being aspirated into the tip. The liquid's density influences the size of the air cushion. Recali- brating pipettors for liquids with substantially different density will improve accuracy. Use reverse pipetting for viscous liquids This technique is used for liquids of high density and viscosity, as well as for easy foaming. Push the piston down to the second stop and draw the liquid up. Then dispense the liquid into the receiving vessel by pressing the operating button gently and steadily to the first stop only. Some liquid will remain in the tip, and this should not be dispensed. The liquid remaining in the tip can be returned to the original source or discarded together with the tip. Reduce pipetting speed and/or use wide bore orifice tips for viscous liquids If pipetting is too fast during aspiration, air bubbles are often formed within the aspirated liquid. Viscous liquids tend to stick to the surface of the tip, and some portion of the pipetted liquid may remain in the pipet tip. The larger the air cushion (e.g., 100 µL dispensed using a 1000 µL tip), the greater the error. The greater the dispenser tip, the lower the dispensable viscosity. Pre-wet at least five times for high vapor pressure or varying temperatures Vapor pressure is a property that describes how fast liquid evaporates into the atmosphere until it reaches equilibrium. All liquids exist in equilibrium between their liquid and gas states. In finding this balance, a liquid will continue to evaporate until a certain concentration is present in its surrounding atmosphere. As soon as the liquid is aspirated into a pipet tip, evaporation begins. The resulting pressure inside the pipettor begins to build and eventually forces some of the liquid back through the tip orifice. Liquids evaporate so quickly that they increase the internal pressure of the air-displacement tips, leading to leaks of the sample. Pre-wetting the tip will help the air space in the tip to reach a concentration closer to the equilibrium point. When a tip is inserted into a warm liquid, the air in the tip is at ambient temperature. During aspiration, the tip heats up which causes the air to expand and push liquid out of the tip. This results in less liquid being delivered than expected. With cold liquids, it has the opposite effect. Pre-wetting the tip will equilibrate the temperature in the tip reducing errors. For additional product or technical information, visitwww.corning.com/lifesciences. product in action Upgrade Your Liquid Handling with Corning® and Falcon® Serological Pipets and Controllers CorningStripettor® Ultra pipet controllers andFalcon pipet controllers offer light, ergonomic, motorized serological pipet control, designed for use with glass and plasticserological pipets from 0.5 to 100 mL volume ranges. Together, Corning and Falcon liquid handling consumables offer a breadth of selection unmatched in the industry with the quality and reliability scientists have trusted for decades. Conveniently positioned switches change operating modes and speeds quickly to handle different liquid volumes and viscosities. Aspirating and dispensing speed is controlled The large LCD display clearly indicates battery status, pipetting mode, and speed. Power docking: Comes with a universal power supply, batteries, and two- (Falcon) or three- (Corning) position charging stands. When fully charged, the batteries enable up to 8 hours of continuous use. Human DNA-free: The serological pipets now feature a human DNA-free claim. Pipets will be tested by DNA detection assay per ISO 18385 and found to be free of detectable human DNA. The limit of detection is 10-11 g of human genomic DNA. Sterility Assurance Level (SAL) of 10-6: The serological pipets are sterilized and dosimetrically released per the requirements of ANSI/AAMI/ISO 11137. The Sterility Assurance Level has been increased from a SAL of 10-3 to 10-6. Quality: RNase-/DNase-free, nonpyrogenic, noncytotoxic, and BSE/TSE-free. Reliability: Negative graduations allow additional working volume. Volumetric accuracy of ±2% at full capacity. To learn more about our lab products, visit: www.corning.com/lifesciences 6 Most modern centrifuges have safety features that, paired with high quality supplies and best practices, protect users and samples. Here are a few tips and things to look for that will help ensure safe operation. CAUTION: The following information is provided to serve as a general guideline for centrifuge use and determining suitability of centrifuge tubes for your applications. In addition, Corning recommends following the procedures outlined by the centrifuge instruction manual, as well as conducting a trial run to determine proper conditions before begin- ning any critical applications. Approved safety containment systems should be used when centrifuging pathogenic organisms, specimens known or suspected of being infectious, or any other potentially infec- tious or hazardous materials. Contact your centrifuge manufacturer for appropriate accessories or recommendations. Confirm temperature stability for tubes The recommended working temperature range forCorning centrifuge tubes is 0oC to 40oC. The suitability of these tubes for storage below 0oC depends on both the solution and the storage conditions. In all cases, tubes that will be frozen should only be filled to 90 percent of the nominal volume indicated on the tube to prevent leaking or damage. In general, the polypropylene (PP) and polyethylene terephthalate (PET) tubes are more resistant to stress at low temperatures than polystyrene (PS). It is strongly recommend- ed that a trial run be performed under actual conditions to test the suitability of the tubes for frozen storage. Do not freeze tubes in polystyrene foam (Styrofoam) racks. The contents hidden under the foam will freeze later due to the difference in thermal conductivity. When liquid freezes from the top of the tube, the force escape area is lost and the tube is much more likely to break when the lower liquid freezes. Confirm chemical compatibility of tubes The mechanical strength, flexibility, color, weight, and dimensional stability of all plastic centrifuge tubes are affected to varying degrees by the chemicals with which they come in contact. Specific operating conditions, especially temperature, relative centrifu- gal force (RCF), rotor type, carrier design, and run length will also affect tube performance. Always conduct a trial run to determine proper conditions before use. Ensure proper balancing and distribution Proper balancing and distribution of the load in a centrifuge is critical for optimum performance and to prevent damage to the tubes or centrifuge. Opposing buckets or loads should always be balanced within the range specified by the manufacturer. Tubes should always be distrib- uted in the buckets with respect to the center of rotation, as well as the pivotal axis of the bucket. Failure to do this may prevent the bucket from achieving a horizontal position during the centrifugation run. Uneven separations or tube failure may result. These centrifuge tubes are intended for use by persons knowledgeable in safe laboratory practices. Failure can result from surface damage, exceeding the RCF values, using unsuitable support systems, improper temperatures, or incompatible chemicals. Read protocols and instruction manuals carefully. Do not confuse speed or revolutions per minute (RPM) with RCF. RCF (relative centrifugal strength) = G (gravity) = 0.00001118 * r * N2 r = Distance from the bottom of the tube to the center of the cen- trifuge (cm) N = RPM (rounds per minute) = Rotation speed Instructions for centrifuging a sample at a given RPM and time are in- complete unless the rotor or radius is specified. Protocols should always state the time and RCF value for centrifuging a sample. Use new tubes every time Centrifuge tubes are single use! We do not recommend the repetition of centrifugation with one tube, as leakage or breakage can occur. Centrifuge tubes can suffer damages during centrifugation such as hairline cracks, deformations, and other compromising damage even if the centrifugation was done within the acceptable conditions. Autoclaving will accentuate any deformation of the tubes and caps caus- ing leakage, contamination, and failure upon further centrifugation. Ensure centrifuge tubes are closed properly To ensure the proper closure and seal between the centrifuge tube cap and tube, we recommend the following procedure: Quick tips Always secure the centrifugation lid for your safety, and make sure the caps are tightly sealed to avoid a mess or biological spill. Mix your centrifuge tube samples by pipetting rather than inversion for cell culture/sterility purposes, as a liquid bridge to exterior when opening can introduce contamination. When marking tubes, black ink Sharpie® pens are the most resistant to alcohol. Other colors tend to smudge. When working in the biosafety cabinet, or simply on the benchtop, "pre-loosening" the caps can save time and frustration. With proper technique, it allows the user to handle tubes with one hand while the other hand may be used to hold the pipettor/pipet controller. Corning® and Falcon® centrifuge tubes are tested for leakage. They should not break or leak if used in a properly balanced rotor with suit- able carriers, holders, and adapters that fully support the tubes when run in accordance with the guidelines in this section. Cell culture is the foundation for many different production and research applications. Culturing cells in controlled conditions gives laboratories insight into tumor development and progression, cell signaling mechanisms, drug sensitivity in specific tissues, and more. Molecular laboratories, cell-based production facilities, and primary research labs all use cell culture, with the basics leading to advanced work with spheroids, organoids, and other multi-cell tissue models. Use appropriate vessels for enhanced culture Factors that optimize cell growth include attachment treatments to encourage, or sometimes discourage, cell attachment on growth surfaces. Most flasks are made of untreated polystyrene, which isn't always optimal for some cell lines. Modifications or coatings make surfaces more hydrophilic to encourage cell attachment and promote cell growth. These coatings include extra- cellular matrix and Corning® BioCoat® treatments with collagen, gelatin, and fibronectin, for example. Advanced options include synthetic, ani- mal-free cultureware, and Ultra-Low Attachment (ULA) surface options. Plan ahead to scale up production Scaling up production is often possible without increasing the footprint. Layering technology using permeable growth surfaces, such as with Corning CellSTACK® vessels or the High Yield PERformance (HYPER) range of products, multiplies the growth surface area for massive scale-up. This technology maintains the product's consistency, which, coupled with ease of use, gives much more cell surface area per footprint at greater efficiencies. Thaw cells gently from storage in liquid nitrogen Passage cells at 85-95 percent confluency Cells in culture require changes in medium to support opti- mal growth. Eventually, the cells multiply so much that they become confluent or tightly packed together. When this happens, you need to split and passage them to avoid mutation, stress, or change in morphology. The passage number is the number of times cells have been split. Frequency usually reflects the cell line doubling time. You can usually assess confluence visually, using direct microscopy for adherent cells. It's also indicated by a phenol red color change. It's best to avoid the culture becoming too confluent as cells may be changed irreparably. Aim to passage cells at moderate to less than 85-95 percent confluency when there's still space to grow. Reliable cryopreservation techniques are crucial for robust experimenta- tion. Cryogenic preservation-storage below 100°C-is used to maintain cell reserves that do not need continuous care. It also provides researchers with backups in case growing cells are lost due to contamination and allows the continuous usage of early passage cells. The success of this operation depends on five things. Keep cultures healthy and harvest gently Prior to freezing, maintain cells in an actively growing state to ensure optimum health and recovery. This can be ensured by passaging the cells or refreshing the growth media one to two days before freezing. Cells should ideally be harvested at a low passage number-this restricts changes to cellular characteristics. It is also important to perform a viability count and check for contamination prior to freezing cells. While harvesting, treat cells gently since it is very difficult for damaged cells to survive the additional stresses that occur during freezing and thawing. Freeze cells in a cryoprotective agent Cryoprotectants are essential to prevent cellular stress during the freeze-thaw process. There are a wide range of chemicals that provide proper cryoprotection. Of these,dimethyl sulfoxide (DMSO) and glycer- ol are the most widely used. DMSO is typically used at a final concentration of five to fifteen per- cent (v/v). Prolonged contact between DMSO and some cell lines can cause adverse effects. A typical solution for this problem is adding the DMSO at 4°C and removing it immediately upon thawing. If that does not suffice, the DMSO concentration can be lowered, or glycerol can be used instead. Glycerol is typically used at a final concentration of five to twenty per- cent (v/v). Glycerol is less toxic to cells when compared to DMSO, but it can cause osmotic problems after thawing. Glycerol should always be added at room temperature or above and slowly removed by dilution. If the cells you work with are particularly sensitive, high serum concen- trations may help with cell survival. Standard medium-cryoprotectant mixtures can be replaced with 95 percent serum and five percent DMSO. Maintain a controlled rate of freezing and temperature consistency Controlling the rate of freezing is important to cell viability. The cooling rate of cultures must be slow enough to allow the cells to hydrate and fast enough to prevent excessive dehydration damage and ice crystal formation. This can be accomplished using a cooling rate of 1°C to 3°C per minute. Cells that are larger or have less permeable membranes may require a slower rate. The best way to control these rates is with an electronic programmable freezing unit. These allow precise control of the freezing process, give uni- form and reproducible results, and can freeze many vials. A less expensive alternative includes using a -80oC freezer with aCorning®CoolCell®con- tainer as a mechanical freezing unit that can control the cooling rate. After freezing overnight, samples can be removed from the unit and transferred to their final storage locations. Store under proper cryogenic conditions For long-term cryogenic storage, keep samples in a freezer that can continually maintain temperatures below -130oC. This can be achieved using most liquid nitrogen cooled freezers and some specially designed mechanical freezers. Most cell culture laboratories prefer liquid nitrogen freezers, but the final choice should be based on the availability and supply of liquid nitrogen, the storage capacity required, and budget. If using a liquid nitrogen freezer, monitor the liquid nitrogen levels regularly to sustain proper temperatures. Store samples incryogenic vials, which are suitable to the low tempera- tures of cryogenic storage, and labeled with inks that can also withstand these temperatures. Labels should be detailed and complete to prevent extended time away from the freezer. Further time savings can be achieved withbarcoded vials, which ensure efficient sample tracking and handling. Thaw cells quickly for optimal recovery After collecting your culture, place the vessel in warm water with agitation. Rapid thawing (60 to 90 seconds at 37oC) typically provides the best recovery and reduces the formation of dam- aging ice crystals. Since some cryoprotective agents can cause damage to cells with prolonged exposure, it is best to remove them quickly and gently through media change or centrifugation. In some cases, about 24 hours after thawing and incubation, cells that appeared viable will have attached loosely or not at all. These cells will either recover slowly or not at all. Somecell culture vessels have specially designed surfaces to increase the chances of cell attachment and survival following cryogenic storage. Endotoxins are a type of complex lipopolysaccharides that perform important structural and functional roles in the outer membrane of most gram-negative bacteria. Bacteria shed endotoxins into their environment in small amounts while actively growing and in large amounts after bacterial death and lysis. These chemical contaminants can have a detrimental influence in vivo and in vitro. There are several common sources of endotoxin contamination in the lab-water, sera, media and additives, glassware, and plasticware- that scientists should be cautious of. Here are a few tips to prevent contamination. Only use high purity water Water is possibly the highest risk material for endotoxin con- tamination. Thus, cell culture laboratories rely on high-purity water for preparing media and solutions, and for washing glassware. Water can be purified using traditional glass distillation, reverse osmo- sis, or by passing it through ion exchange resin and activated carbon columns coupled with a final ultrafiltration treatment. If these systems are not properly maintained, endotoxins can begin to build up. How you store the water after it has been purified is just as important, since bacteria are often found on glass or plastic water storage con- tainers and their tubing. When in doubt, a Limulus amebocyte lysate (LAL) gel clot assay can be performed. If the water or container is an endotoxin source and the problem cannot be resolved,nonpyrogenic high quality water can be purchased. Stick to endotoxin tested sera, media, and reagents Previously, sera-especially in fetal bovine serum (FBS)-have been a major source of endotoxins. The LAL test has increased awareness of these levels, allowing manufacturers to significantly reduce the endotoxin content through aseptic conditions. In addition to stan- dard FBS products, many manufacturers offerpremium certified FBS containing low endotoxin levels (< 0.1 EU/mL). Not all cell cultures are impacted by average amounts of endotoxins, therefore premium FBS is best for researchers that are concerned about issues in their cultures caused by higher contamination. Most commercially prepared media are tested and certified to contain 0.25 EU/mL of endotoxins. This level is typically low enough to avoid adverse effects on cell growth, but any reagents that are added to the media after filtration can also harbor endotoxins-even if they are ster- ile. If in doubt, test the media for endotoxins using the LAL assay both before and after adding reagents. For in-house media preparations, the endotoxin level will be primarily determined by the water used to dissolve other components. Ensure completely sterile glassware and plasticware When using laboratory glassware, it can be difficult to completely remove the strongly adhered endotoxins during washing. Autoclaving glassware using standard procedures has little effect on endotoxin levels due to their high heat stability. To properly destroy contaminating endotoxins all glassware should be subjected to 250oC for more than 30 minutes or 180oC for three hours. When plastic laboratory products are created, the high temperatures used will typically destroy the contaminating endotoxins, but contam- ination can be reintroduced during routine assembly and packaging. The typical processes for plastic sterilization (electron beam or gamma radiation) destroys microbial contaminants and will leave the endotox- ins largely intact. Therefore, it is important to choose plasticware that is certified by the manufacturer to be endotoxin-tested and below a defined level. Ways to Reduce Contamination Risk STICK TO THE BASICS It may seem obvious to some, but don't forget to practice good aseptic techniques and to sterile filter liquids after opening or after adding additives. SHARING IS CARING No one likes having issues with their culture, but it's important to track any problems you have and share them with your lab team. It may help uncover the source of the problem or prevent someone else from running into the same issues. COVER YOUR CULTURE To minimize airborne contaminants, use cell cultureware lids, covers, or caps. TAKE CHANCE OUT OF YOUR PROCESS Re-check all solutions sterilized in-house or stored for long periods of time for contaminants prior to use. Use disposable pipets or aseptic tubing to transfer media and other liquids. Or for more control, use an automated pipet controller that has a sterile filter to minimize the chance of non-sterile air entering the pipet. THINK TIDY It seems like a simple thing, but washing your hands before putting on gloves and using clean lab coats to protect against shedding may help reduce contami- nation risk. Keep your hood clean and uncluttered to maintain airflow and reduce contaminants. Turn off the laminar flow hood only when you'll be away for extended periods. SIMPLICITY IS THE ULTIMATE FORM OF SOPHISTICATION A great way to avoid cross-contamination is by keeping it simple - only work with one cell line and one bottle of media at a time under the hood. To further reduce contamination risk, use small media and buffer volumes and never "double-dip" pipets. ANTIBIOTICS AREN'T ALL THAT Overusing antibiotics leads to an increased chance they will prevent the growth of more easily detected contami- nants but will allow mycoplasma or other cryptic contam- inants to grow undetected. As a result, the cryptically infected culture remains in use and becomes a potential source of serious contamination for the other cultures in the laboratory. Antibiotics are not a substitute for good aseptic technique; however, they can be used strategically as needed in developing, for example, primary cultures. KEEP CALM AND GET ORGANIZED Stay organized and clearly label everything to reduce errors. Another way to reduce confusion is to color-code your work. The lab can also maintain a cell culture log, 15 to document all relevant information for cell lines housed in the lab. TEST YOUR CULTURE OK Test all in-house cell lines to ensure they are free from mycoplasma and other biological contaminants and to check their identity. Test all cell lines that are in continu- ous use at least every 3 to 4 months and any time they behave suspiciously. Better yet: save time, money, and OK effort by periodically discarding these cultures and replacing them with cultures from your tested cell repository. LEARN MORE Visit www.corning.com/lifesciences to watch our on demand three-part webinar series on how to reduce cell culture contamination risk. Want more tips to help prevent contamination? Visit www.corning.com/lifesciences to access webinars, papers, and other related resources. Keep your cells healthy and happy and reduce contamination risk by using Corning® filtration systems. You'll be set-up for cell culture success -- right from the start. Vessels Surfaces Media Use Corning Cell Culture Solutions to Stack the Odds in Your Favor For a listing of trademarks, visit www.corning.com/clstrademarks. All other trademarks included in this document are the property of their respective owners. © 2016 Corning Incorporated. All rights reserved. Printed in USA 8/16 MISC-Contamination Graphic-16 You rely on your lab consumables to perform as promised-to help you deliver consistent, reproducible results at every stage of development. That's why it's so important to choose quality lab products from a partner you can trust. We go beyond supplying laboratory essentials such aspipets,tubes,dishes,flasks,plates, when and where you need them. Corning works with you right from the start to understand your science and your processes. And we provide the expert service and technical support to help you achieve your scientific goals-with confidence.
