Quantibrite Quantibrite Pe Phycoerythrin Fluorescence Quantitation Kit
Additional Materials also see Basic Protocol Phycoerythrin PE Fluorescence Quantitation kit BDB QuantiBRITE PE tubes store up to the expiration date at 2 to 4 C in manufacturer's foil pouch PE-conjugated monoclonal antibody with 1 1 PE protein ratio QuantiQuest v1.0 for Macintosh platforms optional Additional reagents and equipment for direct immunostaining e.g., unit 6.2 1. Remove the QuantiBRITE PE tube from the foil pouch just prior to use. Reconstitute using 0.5 ml PBS BSA azide buffer and...
Info Mpt
Princeton Instruments Binning options Cooling method A-to-D conversion Frame readout time at rate Dynamic range Price SITe SI502FA SIA003AF SITe SI502AB SIA003AB Flexible -30 to -140 C, various methods 14 or 16 bits 0.29 1.2 0.5 sec at 1 MHz 2.32 sec at 500 kHz 84 82 84 88 dB Mid- to high-range Princeton Instruments Binning options Cooling method A-to-D conversion Frame readout time at rate Dynamic range Price Flexible -35 to -60 C, various methods 12 or 14 bits 0.41 1.4 1.4 1.8 4.2 6.3 sec at...
Combined Forward And Side Scatter Detection
The presentation of bivariate data sometimes called cytograms or dot plots of forward versus side scatter has proved to be useful in the flow cytometric analysis of heterogeneous samples. Salzman et al. 1975 demonstrated a crude human white blood cell differential using bivariate displays of forward versus 90 light scattering. The study also reported the first use in flow cytometry of logarithmic amplifiers. The amplifiers were required because of the large dynamic range of the signals. Hoffman...
Multiparametric Immunogating Strategy For The Phenotypic Analysis Of Peripheral
In the example below, the strategy of the LYMPHOGRAM reagent Exalpha Biologicals for three-color stainings is examplified. 1. Display five bivariate plots of the following pairs of parameters forward scatter FS versus side scatter SS side scatter SS versus log green fluorescence CD19-CD8 side scatter SS versus log orange fluorescence CD56-CD3 log green fluorescence CD8-CD19 versus log orange fluorescence CD56-CD3 log green fluorescence CD8-CD19 versus log red fluorescence CD4 . 2. Gate the...
Immunophenotypic Analysis of Peripheral Blood Lymphocytes
Identification, classification, and enumeration of peripheral blood lymphocytes can be performed by several methodological approaches unit 6.2 according to availability of reagents and instrument capabilities. Currently, most commercially available flow cy-tometry instruments have the capability to detect three or four different fluorescence colors. In addition, the vast commercial availability of antibodies directed against lymphocyte-associated antigens, and labeled with various...
Isolation Of Peripheral Blood Monocytes By Gradient Centrifugation With
Peripheral blood monocytes are isolated from blood by centrifugation through a Ficoll-Hypaque or Histopaque gradient Denholm and Wolber, 1991 followed by Percoll. Venous blood in sodium citrate Phosphate-buffered saline appendix 2a 1.077 g ml Ficoll-Hypaque Pharmacia Biotech or Histopaque-1077 Sigma 10x Hanks balanced salt solution HBSS appendix 2a Percoll, specific gravity 1.130 g ml Sigma Tissue culture medium optional appendix 3b 10 x 15-cm round bottom polypropylene tubes silanized with...
Care and Maintenance of Optical Filters
Colored glass filters are relatively robust but should be kept free of fingerprints while in use or storage. Clean with lens tissue as you would eyeglasses and handle around edges. Final cleaning with alcohol on a Q-tip cotton swab followed by wiping with lens tissue will remove most fluorescent materials that might be present. Colored glass filters should be very stable unless used directly in high-intensity light. These filters should be inspected visually for unevenness in color about every...
Info Hqv
full isotype-stained cells are inadequate and in the sample with no CCR5-APC, the isotype-based gate shows 8.8 positive events The difference between a 1-D FMO gate 1.8 and a 2-D FMO gate 5.4 further shows that the multivariate FMO gate will always reveal more of the truly positive events. Finally, Figure 1.14.6C illustrates that FMO gates can even overcome incorrect compensation. In this simplistic example, the goal was to define an appropriate gate to distinguish CD4 T cells. As shown in the...
Characteristics Of Nucleic Acid Stains
A major application of flow and image cytometry is the automated classification of populations of cells on the basis of differences in DNA or RNA content. The DNA content of a cell varies according to its cycle stage of the cell cycle. Tumor cells may or may not exhibit a nuclear DNA content that differs from their euploid normal counterparts. The RNA content of a cell, on the other hand, gives information on whether this particular cell is progressing along a pathway toward a differentiated...
Materials Icx
Premixed monoclonal antibody Cyto-Stat reagents Beckman-Coulter 2-color, 3-color, or 4-color CD45-FITC CD4-RD1 CD3-PC5 and CD45-FITC CD8-RD1 CD3-PC5 CD45-FITC CD4-RD1 CD8-ECD CD3-PC5 Sample of interest whole blood anticoagulated with EDTA ImmunoPrep Reagent System for whole blood lysis Beckman-Coulter ImmunoPrep solution A formic acid ImmunoPrep solution B sodium carbonate sodium chloride sodium sulfate ImmunoPrep solution C paraformaldehyde Flow-Count beads aqueous suspension of microspheres...
DETECTION OF TGase 2 ACTIVATION BY FLUORESCEINATED CADAVERINE FCDV BINDING
This alternate protocol is based on the covalent attachment, by the activated TGase 2, of the fluorescein-tagged cadaverine to the respective protein substrates within the cell Lajemi et al., 1998 . This assay was adapted to flow cytometry and combined with concurrent analysis of cellular DNA content Grabarek et al., 2002 . Like the detergent-based assay, this method is simple and also offers good distinction between apoptotic cells with activated versus nonactivated TGase 2. It should be noted...
Cells and F Reticulocytes in Human Blood
Recent advances in analytical cytometry have improved diagnostic tools for the study of erythropoiesis in anemic patients and resolution of the differential diagnosis in diseases of the erythron. This unit presents three applications of red blood cell RBC analysis quantitation of fetal red cells see Basic Protocol 1 , F cell enumeration see Basic Protocol 2 , and F reticulocyte analysis see Basic Protocol 3 that improve diagnostic precision, sensitivity, and specificity, and provide better...
Hoechst 33258 assay solutions
Stock solution Dissolve in H2O at 1 mg ml. Stable for 6 months at 4 C. Working solution Add 10 ml of 10x TNE buffer see recipe to 90 ml H2O. Filter through a 0.45- m filter, then add 10 l of 1 mg ml Hoechst 33258. Hoechst 33258 is a fluorochrome dye with a molecular weight of624 and a molar extinction coefficient of 4.2 x 104 M 1cm 1 at 338 nm. The dye is added after filtering because it will bind to most filtration membranes. CAUTION Hoechst 33258 is hazardous use appropriate care in handling,...
Choice of Anticoagulant
Although sodium citrate a weak calcium chelator is the most common anticoagulant used for platelet studies, others have been successfully used. EDTA a strong calcium chelator should be avoided, because it causes dissociation of the integrin aIIbp3 GPIIb-IIIa complex. Heparin should also be avoided because it binds to, and may activate, platelets. Nonchelating anticoagulants such as P-PACK a direct thrombin inhibitor may be preferable for the monitoring of GPIIb-IIIa antagonist therapy. The...
Info Six
e.g., house air, pump, compressed nitrogen, etc. . Applications. This plug flow sampling system has several important features that promise to extend the capabilities and usefulness of flow cytometry. First, because sample plugs are of a precisely defined volume 5 l in the present system , particle concentrations are directly determined from the total particle counts in each sample plug. Second, it is not necessary that the vessel containing the sample be pressurized, thus relieving the...
Literature Cited Bxm
Barlogie, B., Raber, M.N., Schumann, J., Johnson, T.S., Drewinko, B., Schwartzendruber, D.E., Goehde, W., Andreeff, M., and Freireich, E.J. 1983. Flow cytometry in clinical cancer research. Cancer Res. 43 3982-3997. Bauer, K.D., Bagwell, C.B., Giaretti, W., Melamed, M.R., Zarbo, R.J., Witzig, T.E., and Rabinovitch, P.S. 1993. Consensus review of the clinical utility of DNA flow cytometry in colorectal cancer. Cytometry 14 486-491. Begg, A.C., McNally, N.J., Shrieve, D.C., and Karcher, H. 1985....
Available Ccd Chips And Cameras
An impressive array of solid-state cameras, incorporating a variety of different CCD chips, is commercially available. These cover a wide range of cost and performance. In this section we tabulate some of the ones that are potentially most useful for cytometry. The list is by no means exhaustive, and the CCD camera situation is subject to rapid change. The performance of a particular CCD camera depends on two major design factors the choice of the CCD sensor itself, and the design of the...
Effects Of Gaussian Laser Beams On Illumination Uniformity
All of the lasers mentioned above except diode lasers emit beams that are radially symmetric, with intensity decreasing with distance from the axis of the beam. The most common intensity profile, associated with an emission mode termed transverse excitation mode TEMoo , is Gaussian in other words, a plot of intensity along any line passing through the axis of the beam would yield a bell-shaped curve identical in shape to the normal or Gaussian distribution frequently encountered in statistics....
Info Kid
for enumerating DCs, as washing could result in selective loss of either cells or counting beads. Regarding immunophenotypical studies for the characterization of each DC subset, the time from sample collection to its technical processing may not be so limiting as for DC enumeration however, it should be noted that at present, information in the literature in this respect is scanty. Thus, processing of samples as soon as possible is recommended for the phenotypic characterization of DC subsets....
Info Koc
Figure 2.6.11 The optical transfer function. A The measured OTF rax 2n in the horizontal direction for a complete system illumination, microscope, scientific, slow-scan CCD camera, and digitizer. B The measured OTFs for a complete system, illumination, microscope, video CCD camera, and digitizer, in both the horizontal and vertical directions. The lens OTF is shown for comparison. cameras a single image frame consists of two interlaced fields representing the odd-numbered lines in one field and...
Materials Jig
DNA from DOP-PCR see Basic Protocol 3 3 M sodium acetate, pH 5.6 unit 8.3 Cells containing chromosomes for hybridization Denaturing solution see recipe prepare fresh 20x SSC, pH 5.3, and 2x SSC, pH 7 appendix2a Dehydration series 70 , 95 , and 100 ethanol, ice-cold Formamide wash solution see recipe 4x SSCT see recipe Blocking solution see recipe prepare fresh 5 g ml fluorescein isothiocyanate-conjugated avidin avidin-FITC Sigma or Vector Laboratories store up to 6 months at -20 C 5 g ml...
Protocol10
Specimen Handling, Storage, and Preparation Handling, Storage, and Preparation of Human Blood Cells bent columns and the magnetic bead separation techniques, can be used in either a positive- or a negative-selection mode. Again, the manner in which these techniques are used depends on the cell surface antigens and their corresponding antibodies, as well as any effect these may have on cell function. Numerous points must be considered in using preenriching or separation techniques prior to flow...
ULTRAVIOLETINDUCED DETECTION OF BrdU IN ETHANOL OR PARAFORMALDEHYDEFIXED CELLS
This protocol describes the ultraviolet-induced detection UVID of BrdU in ethanol- or paraformaldehyde-fixed cells. Exponentially growing cells are incubated with BrdU, harvested, and irradiated with UV-B light to partially photolyze the DNA sections that contain incorporated BrdU and to induce nuclear damage. Cells are then fixed in cold ethanol. Hypotonic conditions subsequently allow the detection of BrdU with monoclonal antibodies. The simultaneous detection of other cellular markers is...
Multicomponent Frap Models
The above methods suffer from the drawback that they cannot solve for two or more diffusing components. If Equation 2.12.7 is written in the form F t F f t , and O is the mobile fraction, the recovery function of the one-component model takes the form F t OF0 f t 1 - O F 0 Equation 2.12.16 and the multi-component model is described by F t a. OiFofi t 1 O. F 0 where a is the fraction of the ith component.
Info Lsi
Any marker of interest can be analyzed using this protocol. Since DCs have an important function as immunomodulatory cells, molecules involved in antigen presentation such as HLA-ABC, HLA-DP, or HLA-DQ , cell adhesion, costimulatory molecules i.e., CD40, CD80, CD86 , immunoglobulin and complement receptors, as well as cytokine receptors including chemokine receptors , can be analyzed. Additional Materials also see Basic Protocol Additional fluorochrome-conjugated mAbs unit 4.2 PE-conjugated...
Chromogenic Detection Using Tsaindirect
Horseradish peroxidase HRP is incorporated into the in situ hybridization product by any of the previously described methods unit 8.4 . The preparation is then incubated in biotinyl-tyramide and hydrogen peroxide H2O2 . In the presence of HRP and H2O2, biotinyl-tyramide becomes covalently bound to proteins proximal to the enzyme site. The reaction occurs via the phenolic group on tyramine, and biotin serves as the label that is subsequently detected. Detection of the covalently bound biotin is...
Titration Of Monoclonal Antibodies To Use With Quantum Simply Cellular
The amount of antibody to be used needs to be optimized. This is done by obtaining the maximum separation of the blank and the first labeled population while having a minimal shift of the blank population to higher fluorescence levels. Verification of the saturation equilibrium can be assessed using small aliquots of Quantum Simply Cellular beads and adding increasing amounts of antibody to them e.g., add additional 50 antibody, until an increase in fluorescence intensity of lt 10 is obtained...
WASHLESS TECHNIQUE FOR BrdU DETECTION
The washless procedure Larsen et al., 1991 Larsen, 1994 combines BrdU labeling with an immunocytochemical BrdU-detection protocol that does not involve repeated centrifugations. Advantages of this approach are its rapidity and the prevention of cell loss that otherwise occurs during centrifugations. The original versions of this procedure, which include washless staining of unfixed nuclei and DNA denaturation by acid, are presented in Larsen et al. 1991 . The method below is a modification of...
Immunofluorescence Assay Using Counterstaining Reagents And Cellquant
A whole-blood specimen or other type of sample is labeled by indirect immunofluores-cence with any primary mouse monoclonal antibodies of the IgG isotype specific for the antigen of interest. An aliquot of the same specimen is also labeled with an irrelevant mouse monoclonal antibody of the same IgG isotype and at the same concentration i.e., a control . A stain no-wash technique is used and the primary monoclonal bound to the cell is detected through a secondary fluorescein-conjugated...
Info Mtd
100 mM magnesium acetate 500 mM potassium acetate 100 mM Trisacetate, pH 7.5 Store at -20 C stable for gt 1 year 1x T4 ligase buffer see recipe 1 mM ATP 8.7 w v glycerol 200 mM NaCl 0.1 g l bovine serum albumin BSA 0.1 g l denatured sonicated salmon sperm DNA 75 nM of each labeled padlock probe see Strategic Planning Sonicated salmon sperm DNA and BSA can be added from 1 mg ml stock solutions, and ATP from a 10 mM stock solution. All are stable gt 1 year at -20 C. Tth ligase buffer, 10x 1 M KCl...
Draq5 Staining Of Fixed Cells
Most commonly used fixation protocols, such as 70 v v ethanol or paraformaldehyde-based methods, appear to be compatible with postfixation staining using DRAQ5. When using fixed samples, consider when to introduce the DRAQ5 staining component. Nuclear-located DRAQ5 is persistent and can be fixed stabilized in live cells and later detected after processing for immunofluorescence analysis, providing a link between the nuclear structures identified in live cells and the location of an...
Diode Lasers
Tens of millions of infrared diode lasers are now sold every year they are incorporated into compact disc players, laser printers, and CD-ROM readers, and now cost only a few dollars each. Red diode lasers emitting anywhere between 630 and 685 nm are also widely used in laser pointers and bar code readers, and are only slightly more expensive. In recent years, red 635 to 640 nm diode lasers have been incorporated into laboratory-built and commercial flow cytometers, usually as an alternative to...
Reagents And Solutions Nqi
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A for suppliers, see SUPPLIERS APPENDIX. 136 mM glucose 75 mM trisodium citrate 4.2 mM citric acid monohydrate Filter through 0.22- .m filter Prepare fresh AES acetic acid ethanol sorbitol fixative Prepare a solution of 18 parts ethanol 3 parts acetic acid 1 part water. Dissolve sorbitol in this solution to a 1 w v final concentration. Prepare fresh. Recipe from Galbraith and Shields 1982...
Otto II buffer
Prepare 0.4 M Na2HPO4- 12H2O. Filter through a 0.22- m filter store up to 6 months at room temperature. Prior to use if stain is to be incorporated directly in the buffer , add 0.1 mg ml DAPI stock solution see recipe to a final concentration of 4 g ml or 1 mg ml PI stock solution to a final concentration of 50 g ml along with 1 mg ml RNase stock solution see recipe to a final concentration of 50 g ml. RNase is needed to prevent PI from binding to RNA. Since DAPI specificially stains DNA RNase...
Basic Protocol 2 Khm
0.1 M Na2B407 10H20, unbuffered pH 9.3 use for nuclei isolated from tissue specimens RNase A solution see recipe 4 formaldehyde Merck in 1x PBS see recipe 0.01 M HCl 0.1 pepsin Sigma in 0.01 M HCl pH 2.0 , 37 C prepare just before use for lymphocytes isolated from blood specimens 1 pepsin Sigma in 0.01 M HCl pH 2.0 , 37 C prepare just before use for nuclei isolated from tissue specimens 10-ml LeucoPrep tubes for blood specimens Becton Dickinson 15-ml polypropylene centrifuge tubes Microtome...
I Hxn
This sequence will allow for the correct location by channel number on the histogram of the normal diploid cells using the diploid control alone. The other samples will demonstrate any slight difference in DNA content as observed by a shift from the channel number obtained in the diploid control alone, and ploidy status is verified through the tissue sample alone. The microscope is an additional analysis tool that is extremely important and often overlooked. Indeed, the microscope is the flow...
Info Ipr
488 nm, Magnesium Green Kd Mg2 of 1.0 mM is analogous to the Ca2 indicator Calcium Green-1. The crown ether chelators used in indicators for Na and K are somewhat less selective than the BAPTA chelator is for Ca2 . Nevertheless, SBFI Minta and Tsien, 1989 has proved to be a valuable indicator for intra-cellular Na , although its spectroscopic response to Na is analogous to the Ca2 indicator fura-2 Grynkiewicz et al., 1985 rather than indo-1 it is therefore not generally suitable for flow...
Materials
Copper block machined to 4 x 4 x 0.75 cm Titanium tubing 5-cm length, V16-in. o.d., 0.01-in. i.d., Upchurch Silicone heat-sink compound e.g., Dow Corning 340, Dow Corning Tellurex Peltier module-based thermoregulation unit Type J thermocouple V16-in. o.d. and 6-in. length, Omega Engineering Watlow Electric Manufacturing model F4 ramping process controller Four Crydom model D1D12 solid-state DC relays Four Newark Electronics model MUR1520 rectifiers, ON semiconductor 2 banana plug to banana plug...
Antih3p Antibodiesprotocol
The method described above see Basic Protocol can be adapted to stain cells mounted on microscope slides for analysis by multiparameter laser scanning cytometry LSC . The cells are initially attached to the slides by cytospinning, then fixed, rinsed, and stained. Additional Materials also see Basic Protocol Tissue culture medium with serum 70 ethanol Paraffin or gelatin-based sealer for coverslips Shandon Cytospin cytocentrifuge and Cytospin chambers Coplin jars Humid chamber vessel with lid,...
DRAQ5 Labeling of Nuclear DNA in Live and Fixed Cells
This unit describes the use of a novel DNA-detecting far-red-fluorescing dye, DRAQ5, which has a unique combination of properties exploitable within live- and fixed-cell cytometry. Excitation at 647 nm, close to the wavelength of maximum fluorescence excitation ExXmax for DRAQ5, produces a fluorescence emission spectrum extending from 665 nm out to beyond 780 nm. Thus the emission spectrum is beyond that of fluorescein, phycoerythrin, Texas Red, Cy3, and perhaps most importantly enhanced green...
Info Tog
Binning options Cooling method A-to-D conversion Frame readout time at rate Dynamic range Price Web site Binning options Cooling method A-to-D conversion Frame readout time at rate Dynamic range Price KAF0400 KAF1400 KAF1600 Arbitrary M x N 10 C, thermoelectric 12 bits 0.41 1.39 1.6 sec at 1 MHz 72.5 67 72 dB Mid-range -25 C, forced air -35 C, liquid circulation 12 bits Photometrics Binning options Cooling method A-to-D conversion Frame readout time at rate Dynamic range Price
Principles of Gating
Gating is an integral part of any flow cytometric analysis. From the discriminator or threshold circuitry used to prevent acquisition of low-level noise signals up through on-line and off-line gating to select specific subpopulations of cells or particles to be analyzed, the different levels of gating available in a flow cytometer provide a means to enhance and simplify analysis of heterogeneous cell or particle populations. It is becoming uncommon for any flow cytometry analysis requiring two...
Frozen Tissue Sections
In this protocol, frozen tissue sections are mildly fixed in formaldehyde by using a short fixation time e.g., lt 15 min , as a means of reducing autofluorescence see Strategic Planning . Proteolytic digestion is tuned so that the cells do not lose nuclear morphology through overdigestion. Compared to Basic Protocol 2, this protocol includes an extra acid dehydration step to avoid relaxation of the nuclei in the tissue section, which results in a loss of nuclear morphology. An optional fixation...
Th7896m
-25 C, forced air -40 C, liquid circulation -90 C, -110 C, LN2 2.8 8.7 sec at 500 kHz 1.4 5.4 5.4 1.4 5.5 5.5 sec at 200 kHz Photometrics Binning options Cooling method A-to-D conversion Frame readout time at rate Dynamic range Price 0.16 0.58 0.74 2.6 3.9 sec at 2 mHz 1.2 1.4 sec at 1 MHz Princeton Instruments CCD-576E CCD-770E CCD-1242E Binning options Cooling method A-to-D conversion Frame readout time at rate Dynamic range Price Web site EEV 02-06 EEV 05-20 EEV 05-30 Flexible -35 to -130 C,...
Info Gma
2. Detach monolayer cultures e.g., using trypsin EDTA and resuspend in complete medium to a concentration of 2-4 x 105 cells ml. Dilute suspension cultures in complete medium to the same concentration. This cell density assumes a human diploid DNA content and an intended staining reaction with 20 J.MDRAQ5. Cells with higher ploidy levels may require higher dye concentrations or lower staining densities. Attached cell cultures e.g., coverslip cultures or chambered wells can be stained in situ...
Amiprophosmethyl treatment solution
Prepare a 20 mM stock solution by dissolving 60.86 mg amiprophos-methyl in 10 ml cold acetone. Store up to 1 year at -20 C in 1-ml aliquots. Prepare the treatment solution immediately before use by combining amiprophos-methyl stock solution and 0.1x or 1x Hoagland's nutrient solution see recipe as specified in Table 5.3.1. Table 5.3.1 Preparation of Amiprophos-Methyl Treatment Solution Table 5.3.1 Preparation of Amiprophos-Methyl Treatment Solution
Materials Esn
Blood to be tested, with tripotassium EDTA or heparin as anticoagulant Phosphate-buffered saline PBS appendix 2a , 10 C Lymphocyte separation medium LSM e.g., Ficoll-Paque PLUS from Pharmacia Biotech, Lymphoprep 1077 or Nycoprep 1077 from Life Technologies, LymphoSep from ICN, Histopaque 1077 from Sigma, or LeucoPREP from Becton Dickinson , 10 C Fluorophore-conjugated monoclonal antibodies directed against the cell surface antigens of interest 15-ml conical polypropylene centrifuge tubes...
Annexin V Binding
Phospholipids of the plasma membrane are asymmetrically distributed between the inner and outer leaflets of the membrane. Phosphatidylcholine and sphingomyelin are exposed on the external leaflet of the lipid bilayer, while phosphatidylserine is located on the inner surface. During apoptosis, this asymmetry is disrupted and phosphatidylserine becomes exposed on the outside surface of the plasma membrane Fadok et al., 1992 Koopman et al., 1994 van Engeland et al., 1998 . Because the...
Resolution
FISH resolution is defined as the smallest genomic distance that must exist between two DNA sequences in order for them to be resolved microscopically. Resolution is inversely proportional to chromatin compaction and is limited by the spatial resolution of the microscope. Apart from the resolution limit, the range over which that resolution is available is a useful specification for probe ordering and mapping by FISH. For metaphase chromosomes, with their highly condensed chromatin, maximum...
Setting Up A Multiphoton Laser Lab
Certain points need to be observed in setting up a multiphoton laser lab. All the mode-locked lasers are sensitive to vibration, dust, and temperature fluctuations. The cleaning of laser mirrors, preferably by means of lens tissue moistened with dry, high-grade methanol, cannot be avoided, but will be required less often if a recirculating air filter e.g., Filtaire 200, TAAB Laboratory Equipment is used. The temperature should be kept to limits of 1 C. An anti-vibration table should be used. A...