j4 Vawe TEA eee Waren, 3c awe rfernonac. OPERATING MANUAL SKATRON SEMI AUTOMATIC \CELL HARVESTERS Cat.no. 7020 and 7023 * Dey Wane Daw Surrevas ARE ON SKATRONAS PO. Box 8. 3401 Ller, NorwayOPERATING MANUAL SKATRON SEMI AUTOMATIC CELL HARVESTER Cat.no, 7020 and 7023 PRODUCT DESCRIPTION The line of Skatron Cell Harvesters was developed for harvesting MLC's (Mixed Lymphocyte Cultures) for thymidine incorporation or lymphocyte stimulation assays. There are now 18 models offering the most commonly requested configuration and the application has expanded to include assays using a variety of isotopes, cell types, nerve cells for example, and even particulate suspensions. A list of available configurations appears below. CELL HARVESTER CONFIGURATIONS woot, T WASH AO ETS ‘waste couccron | = eens mo | . : *Semiaviomaic mode!s Cptionat suction units are avaiaotfor usa wih tubes, vals o other fypes of muliel pos. The most complete units allowing harvest with water, TCA or metha~ nol are also available in semi-automatic versions where reproducti bility and comfort are brought by electronic programming. (no. 7020 and 7023). . PRINCIPLES OF OPERATION NOTE: The following descriptions and examples refer to harvesting In a 96 well microplate, but are also applicable for tubes, vials or multiwell plates: Wash fluid is supplied to the Cell Harvester by gravity feed. Vacuum is provided from an outside source. A suction unit is positioned over the microplate so that pairS of tubes project into the wells to be harvested. The inlet tube, longer and smaller in diameter, feeds the wash fluid into the well while the shorterand wider outlet tube simultaneously aspirates the well contents. This unique rinse principle effectively "scrubs" the well with turbulence giving a thorough harvest without scraping. The cells or cellular debris from each well are transferred to individual dises on a glass fiber filtermat. Following this "harvest", the tubes are. flushed with air to clear excess fluid from the tubing and filter discs to avoid carryover. A single handle cam clamps the filtermat in place for harvesting. EQUIPMENT REQUIRED skatron Cell Harvester*: skatron filtermats** skatron filtermat alignment ‘ device* Wash fluid reservoir(s)** Vacuum collection reservoir(s)** Tubing set** vacuum source *Included with Cell Harvester ** Included in Accessory Pack OPTIONAL ACCESSORY Pilterdise’ Transfer System - Provides fast, easy transfer of discs from filtermat to scintillation vials (for use with 12 well Skatron Cell Harvester only). OPERATING CONTROLS (Viewed with" suction Unit at front). Manual models: (Cat.no. from 7000 to 7018). Top: Filtermat ¢lamp handle Right side: Operating switches, 2 or more, marked "Rinse "Hot", "Cold", "TCA", "Water", "Methanol", "Air". (Air is always last). : Connections: Inlet - 1 or more, marked “Inlet”, "Water", "TCA", “Methano. outlet - 1 or 2, marked "outlet" or "Hot" and "Cold". Semi-Automatic Models (Cat.no. 7020 and 7023). Top: Filtermat clamp handle. Operation touch pads - Prewet, Start, Stop. and Control Lamps - Cold, Hot, Dry, Wash 1, 2 & 3. Left side: Wash I, II and III counters. Back:connections 3 inlets marked water, TCA, Methanol. 2 outlets marked Hot and Cold.INSTALLATION Remove Cell Harvester from box. Remove packing around, suction Rome Gnd tape around harvesting blocks. Screw handle into can unit atop of the Cell Harvester. Install filtermat alignment at vice. “Ensert power cord into receptacle on rear of harvestex, gee pO NOT CONNECT TO POWER SOURCE UNTIL TUBING CONNECTIONS ARE COMPLETED. For proper operation, all tubing should have a mininun internal cfaneter of 8 mm. Vacuum tubing must not collapse when vacwun Ge applied. Place wash fluid reservoir(s) 1 - 1,5 m above level is GR0i Harvester. Attach tubing from wash fluid reservoir(s) fo appropriate iniet(s). On single outlet models, connect "outlet" te apprortuum collection reservoir, and connect the reservoirs to a vacuum source. House vacuum or a water driven pump may suffice. If not, a separate vacuum pump (available from skatron) is recommended. For dual outlet models, attach vacuum tubing from the "hot" and weed" outlets to "Hot" and "Cold" vacuum zeservoirs. The reser voirs can be attached by a “¥" connector to the sane vacua vCores. Ifa water driven aspirator is used, it may be possible fo run the "cold" waste directly into the aspirator - CONSULT YOUR RADIATION SAFETY OFFICER FOR LOCAL REGULATIONS REGARDING "HOT" AND "COLD" WASTE DISPOSAL BEFORE USING THE DUAL OUTLET HARVESTER. 7 OPERATION Wash fluid MLC Harvesting can be performed with either distilled water of eS tused tap water. Distilled water can be added directly to the euch fluid reservoir. Tap water should go through a 0,2 mm line We ter into the reservoir, Other wash fluids such as TCA and ethanol can be used depending on the model of Cell Harvester» NOTE: Wherever the wash fluid is designated as water, saline can be used instead if desired. Switch Functions (for manual models no. 7000 to 7018). operating switches are placed in the order in which they will generally be used during harvesting. For dual outles harvesters, genet tree switch will generally channel the waste through the ‘Hot’ outlet. Remaining switches will run the waste through the "Cold" outlet. Specific switch functions are: Rinse - water wash; single outlet. Hot - water was Hot" outlet. cold = water wash; "Cold"outlet. TCA 7 fea wash (usually first); usually “Hot” if dual outlets. Methanol - Methanol wash; usually "Cold" if dual outlets. Water T water wash (in conjunction with other wash fluids); "Cold" if dual outlets.Control panel _and Counters (for semi-automatic no, 7020 and 7023) Prewet: (wash I) flushes water during 2 seconds. Start: can only be actionned if Prewet has been run first ~ each wash (1 - 2 - 3) will be operated one after the other, non stop. (if the counter of a wash is set at zero, the wash will not be run). During the first six seconds, all waste will go through the "Hot" outlet. The rest of the waste will go through “Cold”. After the wash routine, filters will be dried during 10 seconds. Stop: Indicates that the program is finished. Can be used to stop the program at any time. Wash time counters - Three, one per wash. 10 position digital gaitGles which control the duration of the washes. The range is from 0 - 9 where0 indicates that the wash will not be performed and 9 indicates the longest time. These are index numbers only and do not represent actual time units. Refer to the equivalence sheet. Harvesting blocks - The blocks clamp the filter in place during flarvesting, Depending on the mode!, there will be either 6 or 12 holes. The lower block contains O-rings which seal off discrete dises on the filtermat. Holes in the upper block are covered with screens to support the disc during harvest. The fluid flow @irection during harvesting is up through the blocks. A special venturi design spreads the liguid stream to utilize the entire ise surface. Waste fluid is pooled in the upper. block and exits through an outlet in the rear on the right side of the block. The pattern of wells harvested is shown below. Only positions 1-6 would apply for § well models. There are two black dots on the front of the upper block to assist in positioning the filtermat. Filterdise capacity - The 12 well Skatron Cell Harvester has a Fe eras sm giving a capacity of 2 million cells per filtedisc. If a greater capacity is needed, the 6 well Large Harvest models offer 16 mn diameter discs with a capacity of lo million cells per filterdisc. ©®suction Unit - The suction unit is silicone rubber with sets of Stainless steel tubes specifically designed for the plate, tube, or vial being used. The Skatron Cell Harvester comes with a suc~ tion unit for round bottom 96 well microplates as standard equip~ ment. Suction units are also available for flat bottom micro- plates, 12 and 24 well plates, and a variety of tubes and vials. Baction units are easily interchangeable by simply pulling off the 12 numbered tubes and the large supply tube and re-attaching them on a different suction unit. Filtermat - The filtermat is made of binder-free glass fiber with | particle retention of at least 1,5 um. Thickness and fluid flow Gharacteristics are incorporated into the overall design of the Cell Harvester. To insure proper performance, it is recommended that Skatron-supplied filtermats be used. whe £iltermat has 8 notches on each side to facilitate proper alignment. These create 8 equal harvesting areas plus a 1 cm tab upon which an identification can be marked. The filtermat should aiways be insterted tab first throught the rear of the harvester blocks. If the filtermathas a noticeable rough side, it should be down. By, following this procedure, ‘the “A-1" well will always be at the left edge of the identification tab. NOTE: The top sheet in each box of Skatron filtermats is for pro tection only. IT DOES NOT HAVE ALIGNMENT NOTCHES AND IS NOT A FILTERMAT. Before Harvesting Check the Following ~ Mains cable (power cord) is connected to an operating circuit. ~ There is sufficient wash fluid. = any valves in tubing or reservoir are open. = yacuum Collection reservoir has adequate capacity. = Suction unit is operating properly. ~ Cell Harvester valves are operating properly- SUCTION UNIT CHECK Inlet tubes- With all valves in the wash fluid tubing and reser- voir open, hold the suction unit over a sink or pail and press the "rinse" switch (or "cold" or “water"). After any air has cleared from the tubing, there should be 12 uniform streams of fluid coming from the inlet (longer) tubes. Uneven or poor flow in any of the stream indicates a blockage. See the “Trouble Shooting" section for corrective action. outlet tubes - Turn vacuum on. Without a filtermat in place, pull the clamp handle forward to close the blocks. Insert the suction unit in a row of wells on a microplate. Press the “rinse” (or “cold” or “water") switch. Observe the 12 tubes leading from the suction unit into the front of the Cell Harvester for uniform flow. See "Trouble Shooting" for corrective action.CELL HARVESTER VALVE CHECK Plug Cell Harvester into an operating electrical outlet. With vacuum off and valves closed in wash fluid reservoirs, press each operating switch. A distinct, "click" will be heard as each switch is depressed if the valves are operating properly.TO HARVEST A MICROPLATE Turn vacuum on. Insert a filtermat, tab end first and rough side down through the rear of the blocks and inside the clamp of the filtermat alignment device. Place the clamp of the alignment device into the first hole of the guide bar. Clamp the f£iltermat in place by rotating the filter clamp handle toward the front of the harvester. Insert the suction unit into a row of wells on a clean micro- plate. For manual models: Operate the "rinse switch (or "col "water") for 2 - 3 seconds. This prewets the filter. For automatic models: Depress “Prewet". Move the suction unit to the "A" row of the microplate to be harvested. For manual models: Single outlet harvester ~ Operate “Rinse” for 20 Seconds, then "air" for 10 seconds. Dual outlet harvester - Operate "Hot" or first wash switch for about 5 seconds: (This collects 90 - 958 of the unbound isotope in the “Hot* vacuum collection reservoir). Operate "Cold" or additional wash switches for at least 15 seconds; then operate "air" for 10 seconds. For automatic model - Preset the duration of each wash by setting the wash counters at the proper value. (If set at 0, the corresponding wash will not be performed ). - Depress "Start". During the first 6 seconds, the waste will be sent through the "Hot" outlet. The rest will be collected through the "Cold" outlet. Each wash will be performed after each other. The cycle ends by a 10 second drying sequence. Unclamp the filtermat. Gently pull it down and advance it to the next set of samples. (Next hole of alignment device). Before moving the suction unit to the next row Operate the "rinse" switch (manual models) or "Prewet® (auto- matic models). The Skatron Cell Harvester is so effective that the row of wells just harvested is clean enough for the prewetting step.8. “Repeat steps 5 - 7 until the microplate has been completely harvested. 9. Remove the filtermat and dry at 60°C for at least 60 minutes. 10. Punch out filterdiscs and transfer to scintillation counting vials. (This may be performed individually with forceps or 6 at a time with the Skatron Filterdisc Transfer system) . 11. Add approximately 2 ml scintillation fluid to each vial and count. PREVENTIVE MAINTENANCE IMPORTANT - When saline is used as wash fluid, the Cell Harvester should be flushed with distilled water or filtered tap water at the end of each work day. This prevents corrosion’ and the buildup of crystalline precipitates. The glass fiber filtermats leave a fine white dust on the O-rings of the lower harvesting block. This should be cleaned DAILY with a paper towel or soft brush to prolong O-ring life. The upper harvesting block slides up and down on two vertical shafts. These should be lightly lubricated with silicone grease after every 10 hours of operation. TROUBLE SHOOTING Problem - Harvester will not operate. Solution ~ Check that electrical outlet is operating. Check fuse. (A spare fuse is taped to the underside of the Cell Harvester). Problem - Blocked inlet tube on suction unit. Solution - Insert a hypodermic needle or narrow wire into the blocked inlet tube. If this fails, remove the inlet supply tubing from the right side of the suction unit and connect the suction unit to a vacuum source. With the vacuum on, again insert hypodermic needle or narrow wire. Problem ~ Blocked outlet tube on suction unit. Solution - Insert a hypodermic needle or narrow wire into the blocked tube. If this fails, remove the outlet tubing from the back of the suction unit. Insert a tight fitting hypodermic needle on a syringe and blow air into the tube until the blockage is cleared.Problem - Solution - Flooding of 12 wells. (Por flooding of a single well, see "Blocked Outlet Tube on Suction Unit"). Check that vacuum is on. Check for vacuum leaks or blockages in tubing. Check that vacuum collection reservoir is not full. Check outlet valve by pressing "air" switch. Increase vacuum strength. Problem ~ No wash fluid in all 12 wells. Filterdiscs will be dry. (For no wash fluid in a single well, see Locked Inlet Tube on Suction Unit"). Solution - Problem — Solution - Problem - Solution ~ Problem - Solution - Check that there is adequate wash fluid in reservoir. Check that all valves in reservoir or inlet line are open. Be sure that there is no air in the inlet tubing. Check inlet valve. A wash will not be performed. Check that the corresponding counter is not set at 0. Dises are cut out of filtermat ‘by blocks. Check that Skatron filtermats are used. Check that O-rings are clean. Dises do not separate after harvesting. Check that Skatron filtermats are used. Check that O-rings are intact. Copyright skatron 2/S, Lier, Norway 1984