Guidelines for Bioassays on MultiScreen Filter Plates: Protein Binding and Cell Loading

The MultiScreenHTS 96-well filter plate

Optimization Parameters for Receptor Binding, Enzyme Activity, Drug Discovery and Related Assays

The MultiScreen Assay System is well suited for a variety of separation techniques, including cell separations and solid phase bioassays. The MultiScreen system makes it possible to carry out an entire assay from sample incubation to scintillation counting in a single 96-well plate. The advantages of the system include a 96 % or more reduction in radioactive waste over traditional cell harvesting methods, improved coefficients of variation, better assay reproducibility, and faster multiple sample processing. Most assays carried out using historical methods and techniques can now be carried out on one of the different filter media available on the MultiScreen 96-well plates. To ensure accurate, reliable assays, certain technical parameters should be observed depending on the type of MultiScreen plate you select. These issues are particularly important when “miniaturizing” an assay, or when optimizing an established method with the actual 96-well plate. These guidelines will help you select the optimal filter for a specific application.

For assays where binding capacity required exceeds that of membranes, the Durapore^® plates loaded with suitable chromatography media are recommended. Use of plates containing glass fiber or filter papers is not recommended when adding resins or beads. Increased dead volume, higher non-specific binding and sometimes reduced filtration capacity are caused by interactions between filter materials and media. Plates are available for both solvent-based and aqueous applications.

In addition to selecting the right membrane material for your assay, you must also make sure to select the right plate material. Table 1 for recommendations.

Table 1. Plate Type (Material) and Recommended Uses

Plate Type	Characteristics/Uses
Clea	General use (aqueous buffers,etc.) Up to 70 % DMSO, < 10 % org
Opaque	Aqueous buffers, etc. (microplate scintillation counting, flash chemiluminescence, no strong chaotropes)
Solvinert	Solvent-compatible
Black	Cell culture solutions (Fluorescence detection)

In some of the following tables, not all of the available MultiScreen membrane filters will have data, since some filters are inappropriate for certain techniques. The five technical parameters addressed are: 1) plate preparation, 2) protein binding, 3) protein (IgG) loading or membrane capacity, 4) cell loading, and 5) bacteria loading.

Materials and Methods for Guidelines Testing

Methods for determining experimental parameters for the different MultiScreen plates are described in the appropriate sections. The materials listed below were used in the various studies. Proteins and ¹²⁵I labeled proteins were purchased from Sigma Chemical Co. (St. Louis, MO), ICN (Irvine, CA) and NEN (Boston, MA).

Table 2. Materials and Methods for Guidelines Testing

Protein	Supplier	Catalog No.
Bovine serum albumin (BSA)	Sigma	A-4503
Bovine γ-globulin (IgG)	Sigma	G-7516
Porcine insulin	Sigma	I-5523
125I-BSA	ICN	68031.2
125I-IgG	NEN	NEX-155
125I-Insulin	ICN	6804410

Solvent-resistant MultiScreenHTS vacuum manifold

MultiScreen Plate Preparation and Punching Recommendations

Prior to the addition of assay reagents, the individual wells of the MultiScreen plate may require pre-wetting for optimal assay performance. Table 3, column 3, shows the recommended pre-wet. To pre-wet the plate, add appropriate liquid, incubate for one minute, filter through using the MultiScreen or other vacuum manifold. Some membrane types can be punched into scintillation or gamma vials using the MultiScreen Multiple Punch. Table 3, column 4, shows punching capability. MultiScreen opaque plates are recommended for direct detection with microplate scintillation or luminescent counters.

Table 3. Preparation Notes for MultiScreen Plates

MultiScreen Plate	Pore Size/Membrane	Pre-Wet	Punchable
GV	0.2 µM Durapore	Yes	Yes
HV	0.45 µM Durapore	Yes	Yes
DV	0.65 µM Durapore	Yes	Yes
BV	1.2 µM Durapore	Yes	Yes
RL	0.4 µM Philic Solvinert	Yes	No
RP	0.4 µM Phobic Solvinert	Yes (90 % ethanol)	No
IP	0.45 µM Immobilon™-P	Yes (50 % ethanol)	Yes
HA	0.45 µM MCE	Yes	Only damp
FC	Glass Fiber, Type C	Yes	Yes
FB	Glass Fiber, Type B	Yes	Yes
DE	DEAE	Yes	Only damp
PH	Phosphocellulose	Yes	Only damp

Protein Binding Guidelines

Protein binding levels vary directly with the type of membrane media in the MultiScreen plate1. For high protein binding applications use IP, HA, DE, or PH plates. For high nucleic acid binding use HA, FB, FC, or DE plates. For low biomolecule binding use Durapore plates. For assays that require solvent resistance, use RL or RP Solvinert filter plates. The FL plates are recommended solely for cell culture assays. General guidelines for protein binding are reported in Table 4.

Table 4. Protein Binding Capacity of MultiScreen Plates

MultiScreen Plate	Pore Size/Membrane	BSA (µg/well)	IgG (µg/well)	Insulin (µg/well)
GV	0.2 µM Durapore	< 1.5	< 1.5	< 1.5
HV	0.45 µM Durapore	< 1.5	< 1.5	< 1.5
DV	0.65 µM Durapore	< 1.5	< 1.5	< 1.5
BV	1.2 µM Durapore	< 1.5	< 1.5	< 1.5
FL	0.4 µM PCF	< 10	< 10	< 10
IP	0.45 µM Immobilon-P	25	47	26
HA	0.45 µM MCE	36	34	18
FC	Glass Fiber, Type C	31	21	6.0
FB	Glass Fiber, Type B	38	27	6.0
DE	DEAE	29	19	47
DE (KPO/pH 7.51)	DEAE	35	ND2	ND2
PH	Phosphocellulose	3.2	10	3.8
PH (NaAc/pH 4.51)	Phosphocellulose	20	ND2	ND2

¹Protein eluted in 0.5 M NaCl for total protein assay
²ND = Not determined

The MultiScreen-FC and FB glass fiber filter plates show high binding capacity for BSA and IgG, but low binding capacity for insulin. These plates are primarily intended for receptor binding assays, particularly with crude receptor preparations. The FB plates are also suitable for DNA binding.

The plates containing Durapore membranes, including GV, HV, DV and BV, all demonstrate low protein binding. These membranes have excellent nonspecific binding levels (NSB) and are recommended for column separation assays, enzyme assays, and biological sample preparation in aqueous conditions. These MultiScreen plates are used extensively for cytokines such as interleukins^{2,4,5 and 6}. They are also ideal for filtering multiple small volume samples directly into plastic 96-well plates.

Each microporous membrane interacts distinctly with each protein. For this reason, it may be necessary to test different membrane types for each assay. The MultiScreen Assay System Applications and References Guide (RF016) recommends plates by assay type, and lists references to published applications.

Procedure

The following procedure was used to determine protein binding levels for various media:

Plates (with the exception of IP) were pre-wet with 200 µL phosphate buffered saline with 0.01 % sodium azide, vacuum drained, and then the underdrain was blotted on clean paper towels. The IP plate was first pre-wet with 20 µL of 50 % ethanol, vacuum drained, and then washed three times with 200 µL PBS and blotted on paper towels.

Using a multichannel pipette,100 µL of ¹²⁵I-BSA, ¹²⁵I-IgG or ¹²⁵I-insulin was added into one row (8 wells) on each plate. Each ¹²⁵I-labeled protein solution was a 1 mg/mL solution with approximately 10⁵ CPM per 100 µL. Plates were incubated for two hours at room temperature, vacuum drained, washed and vacuum drained four times with 200 µL of PBS, and blotted on paper towels. The underdrain was removed and the membrane blotted on paper towels. Plates were punched using the MultiScreen punch assembly into 12 x 75 mm test tubes.

Gamma counting was carried out on each membrane using Packard’s Minaxi-γ Auto-Gamma^® 5000 Series.

Protein Loading Recommendations

For optimum assay sensitivity, the loading levels of protein for each well of the MultiScreen plate should be kept at a level that maximizes effective washing. The loading guidelines noted in Table 4 below indicate general protein loading levels based on studies of TCA-precipitated IgG. The criteria for recommended maximum loading was the capacity to perform each wash at 12" Hg vacuum in less than 20 seconds.

To determine protein loading levels, plates were first pre-wet, as in Table 3. The first column of the plate was loaded with a 10 % IgG solution and serial diluted in PBS across the plates to achieve a range of dilutions. Next, 100 µL of cold 20 % trichloroacetic acid (TCA) was added to all wells on all plates. Plates were incubated at 4 °C for 30 minutes and vacuum drained. Plates were then washed three times with 10 % cold TCA.

Cell Loading Recommendations

Cell loading studies were performed on the MultiScreen plates to determine the maximum level of cells per well that can be filtered and washed efficiently. These recommendations are useful for cell-based assays involving crude receptor preparations or cell culture suspensions. The recommended guidelines for cell loading on appropriate MultiScreen media are shown in Table 5. Cell loading studies on the various MultiScreen plates were carried out with mouse hybridomas (ATCC #TIB-111). Cells were grown and resuspended to a final concentration of 5 x 10⁶ cells/150 µL. After pre-wetting the plate, the first column was loaded with 150 µL of sample.

Serial dilutions were made in 150 µL of media across the plate to achieve desired cell loads. After incubation, vacuum was applied and the plates washed three times with PBS. Criteria for maximum recommended loading was the ability to accomplish each of the three washes at 12" Hg vacuum in less than 20 seconds. Recommendations for receptor preparations are based on this data and filtration capacities for the membranes.

Table 5. Guidelines for Protein Loading Determined by TCA Precipitation

MultiScreen Plate	Pore Size/Membrane	Maximum Recommended Protein/Well
GV	0.22 µM Durapore	100 µg
HV	0.45 µM Durapore	100 µg
DV	0.65 µM Durapore	100 µg
BV	1.2 µM Durapore	600 µg
HA	0.45 µM MCE	200–300 µg
FC	Glass Fiber, Type C	1.25 mg
FB	Glass Fiber, Type B	1.25 mg
RL	0.4 µM hydrophilic PTFE	40 µg

Seeding Density Recommendations

Seeding density is the recommended number of cells/well to load onto a plate for cell culture, e.g. for subsequent cell proliferation assays.

Table 6. Guidelines for Cell and Cell Fragment Loading of MultiScreen Plates

MultiScreen Plate	Pore Size/Membrane	Cell Loading1	Maximum Receptor
GV	0.22 µM Durapore	2 x 105	Purified only
HV	0.45 µM Durapore	2 x 105	(< 30 µg T.P.)
DV	0.65 µM Durapore	2 x 105	< 30 µg, purified
BV	1.2 µM Durapore	6 x 105	> 30 µg “Semi-purified”
HA	0.45 µM MCE	3–6 x 105	NR2
FC	Glass Fiber, Type C	2.5 x 106	Crude receptors
FB	Glass Fiber, Type B	2.5 x 106	(homogenates)

¹Mouse hybridoma cells
²NR = Not recommended

Bacteria Loading Recommendations

For sample preparation of bacteria, a procedure that maximizes effective washing will maximize assay performance. The test was conducted with suspensions of E. coli JM109 (Stratagene, La Jolla, CA) in 2X LuriaBertani (LB) broth, grown to stationary phase at 37 °C, in an orbital shaker, (15–8 hours). The cell density was calculated by reading the OD 600 nm (in a Beckman DU-640 spectrophotometer) of the suspension⁷, and diluted as needed. The criteria for recommended maximum loading was the capacity to perform three washes in less than 60 seconds.

Table 7. Guidelines for E. coli Loading of MultiScreen Plates

MultiScreen Plate	Pore Size/Membrane	E. coli load (x 107 cells/well)
GV	0.22 µM Durapore	10
HV	0.45 µM Durapore	10
DV	0.65 µM Durapore	10
BV	1.2 µM Durapore	20
FL	0.4 µM PCF	5
IP	0.45 µM Immobilon-P	10
HA	0.45 µM MCE	20
FC	Glass Fiber, Type C	> 20
FB	Glass Fiber, Type B	> 20
DE1	DEAE	12
PH1	Phosphocellulose	6

¹Run at recommended 6" Hg Vacuum instead of 12" Hg.

Before adding the cell suspension, all the wells were pre-wet by adding 200 µL of PBS, then vacuum draining. The first column of each plate was loaded with stock suspension and serial diluted in 2X LB across the plate in rows of 8 wells. The plates were then vacuum drained and washed three times with 200 µL of PBS. All the wells were then washed with PBS (3 x 200 µL/well) and filtered under vacuum (12" Hg).

Summary

For optimum results, it is important to select the most appropriate plate based on binding and loading capacity, and to pre-wet as recommended. For detailed procedures and recommendations for specific assays.

References

1. Pitt, A. 1987. The Non-specific Protein Binding of Polymeric Microporous Membranes. . J. Parenteral Sci. & Technol. . 41, 110-113..

2. Cain, M. 1993 . Cloned Human Interleukin-1 Receptor (Type 1) with [125I]-Interleukin-1a Provides Convenient System for Screening or Competition Studies. . J. NIH Research. . 5, 81..

3. Slack, J, Sims, J, Pitt, A, Dower, S. 1989 . Application of the MultiScreen System to Cytokine Radioreceptor Assays. . Biotechniques. . 7, 1132-1138..

4. Horuk R. 1989. A rapid and direct method for the detection and quantification of interleukin-1 receptors using 96 well filtration plates. Journal of Immunological Methods. 119(2):255-258. https://doi.org/10.1016/0022-1759(89)90404-3

5. Anthes JC, Zhan Z, Gilchrest H, Egan RW, Siegel MI, Billah MM. 1995. Interferon-? down-regulates the interleukin-6 receptor in a human multiple myeloma cell line, U266. 309(1):175-180. https://doi.org/10.1042/bj3090175

6. Lusti-Narasimhan M, Power CA, Allet B, Alouani S, Bacon KB, Mermod J, Proudfoot AE, Wells TN. 1995. Mutation of Leu25 and Val27 Introduces CC Chemokine Activity into Interleukin-8. Journal of Biological Chemistry. 270(6):2716-2721. https://doi.org/10.1074/jbc.270.6.2716

7. Maniatis, T, Fritsch E, Sambrook, J. 1989. Molecular Cloning: A Laboratory Manual, . 2nd ed., p. 61.. Cold Spring Harbor, NY, : Cold Spring Harbor Laboratory Press, .