SpectraMax ABS Plus bench manual

0 · Before you start

Five minutes of reading here will save you a ruined plate later.

The two sample ports

Confirmed as a SpectraMax ABS Plus — serial prefix ABP, cuvette lid on the top centre, plate drawer at the front bottom. Everything in this manual matches that layout.

That difference in light path is the reason the two halves of this manual diverge. A cuvette reading goes straight into Beer–Lambert. A plate reading does not, unless you correct it — see 2.5.

Safety and handling

• Wear gloves. Handle cuvettes by the frosted sides only — fingerprints on the clear windows are a real source of baseline error.
• Plate height must not exceed 17 mm including the lid. A taller plate will jam the drawer.
• Organic solvents can damage the optics. Keep plates lidded and don't leave solvent-containing samples sitting in the chamber.
• If you spill inside the drawer, power off, wipe with a lint-free absorbent wipe, and let it air dry fully before powering on again.

What the instrument can do

Cuvette reads take about 1 second for a single-wavelength endpoint. A full 96-well plate takes about 9 seconds at one wavelength, 19 seconds at two.

A · Power-on and connect

Do this once at the start of a session, whether you're reading a cuvette or a plate.

1. Check that the plate drawer and cuvette chamber are empty and the cuvette lid is closed. Leaving the cuvette lid open during start-up causes stray-light errors.
2. Switch the power switch on the rear of the instrument to On. Wait for the diagnostic check to finish and the plate drawer to open. This takes under a minute.
3. Power on the computer and launch SoftMax Pro from the desktop icon, or Start › Molecular Devices › SoftMax Pro 7.1.2.
4. On the Home tab of the ribbon, click Instrument. In the Instrument Connection dialog, select the USB port the reader is on and click OK. Click Refresh first if nothing is listed.
   • The instrument icon on the Home tab shows the connection state. If it shows disconnected, check the USB cable and that the reader finished its start-up.
5. If you need temperature control, click Temperature on the Home tab and set the plate or cuvette chamber. Allow 15–30 minutes to reach setpoint.
   • The setpoint must be at least 5 °C above ambient. There is no cooling — only heaters.
6. Save the document before you collect anything: Application menu › Save As, choose Data Files (*.sda), and give it a real name. Working in an unsaved Untitled1 is how data gets lost.

1.1 Choosing and filling a cuvette

The cuvette chamber accepts standard 45 mm cuvettes and taller ones up to 56 mm, with a 10 mm pathlength and 12.5 × 12.5 mm outer dimensions. It does not accept short 25 mm microcuvettes.

The light beam passes 15.87 mm above the bottom of the cuvette. Your meniscus must sit comfortably above that, or you will be measuring air.

Fill the cuvette, cap or cover it if the sample is volatile, and wipe the two clear windows with a lint-free tissue. Check for bubbles against the light — a bubble in the beam path is the most common cause of an absurd reading.

1.2 Set up the read in SoftMax Pro

Cuvette data lives in a Cuvette Set section, which is a different section type from a Plate section. A Cuvette Set holds a series of cuvette readings labelled A1, A2, A3 and so on.

1. In the Navigation Tree on the left, select the section you want the new one to sit below.
2. Click New Cuvette Set. A Cuvette Set section appears in the workspace.
3. Right-click the section and choose Rename to give it something meaningful — the name carries through to the exported file.
4. With the Cuvette Set section selected, click Acquisition Settings (bottom of the workspace, or on the Home tab) to open the Settings dialog.
5. Choose your read type and wavelengths:
   • Endpoint — enter up to six wavelengths. Enter them in ascending order.
   • Spectral Scan — set start and end wavelength and the step interval. Cuvette scans run at roughly 45 nm/min per nm of interval at normal speed, 130 nm/min at speed read.
   • Kinetic — set total run time and interval. The minimum cuvette interval is 2 seconds at a single wavelength.
6. Under the reduction settings, choose whether you want Optical Density (OD) or %Transmittance. OD is what you want in almost every case.
7. Click OK and save the document.

1.3 Blanking and the reference read

There are two different things in SoftMax Pro that both sound like "reference", and confusing them is a common source of trouble.

For a routine cuvette measurement:

1. Fill a clean cuvette with your blank — the same buffer or solvent as your sample, minus the analyte. Never blank against an empty cuvette.
2. Load it (see 1.4) and click Ref in the Cuvette Set section toolbar.
3. Remove the blank, rinse the cuvette with your sample or use a matched second cuvette, and proceed to the sample read.

If you skip the Ref read, the instrument falls back to the I₀ values stored in its firmware. That is acceptable for rough work but you should blank properly for anything you intend to publish.

Alternatively, read the blank as its own cuvette in the set and subtract it during analysis. This has the advantage of keeping the blank value visible in the exported data, which reviewers like.

1.4 Load the cuvette and read

1. Press down on the cuvette chamber lid on the top centre of the instrument to open it.
2. Drop the cuvette in. Springs in the chamber hold it in alignment. Orient it so the clear windows face front and rear — the beam runs front to back, not left to right.
3. Close the cuvette lid by hand. It must be closed before you read.
4. Select the Cuvette Set section in the Navigation Tree and click Read on the Home tab.
5. The reading appears in the section as cuvette A1. For the next sample, swap the cuvette and click Read again — it lands in A2, and so on.
6. Save the document.

1.5 Common cuvette measurements

When you're finished, go to section 3 to get the data into Excel.

2.1 Choosing a plate and volume

• Use flat-bottomed plates with isolated wells. Round-bottom plates work but give less consistent optics.
• Fill volume of 100–300 µL per well in a standard 96-well plate. This is the range in which PathCheck-corrected plate values agree with cuvette values.
• Plate height including lid must not exceed 17 mm.
• If you're incubating, use a lid or seal. Evaporation changes the pathlength mid-run and will corrupt a kinetic read.

2.2 Set up the read in SoftMax Pro

1. In the Navigation Tree, click New Plate. A Plate section appears. Rename it via right-click if you want a meaningful label in the export.
2. Optionally use the Protocol Manager on the Home tab to start from one of the built-in protocols instead. There are over 160, including standard nucleic acid and protein assays, and they arrive with sensible reduction formulas already set.
3. With the Plate section selected, open Acquisition Settings.
4. Set the plate type to 96-well and confirm the read area if you're only using part of the plate. Restricting the read area saves time on partial plates.
5. Choose the read type and wavelengths:
   • Endpoint — up to six wavelengths, entered in ascending order.
   • Kinetic — minimum interval is 8 seconds for a full 96-well plate at one wavelength, or 3 seconds if you restrict the read to a single column.
   • Spectral Scan — normal scan runs at 33 nm/min per nm of interval for 8-well strips.
6. Set Shake if the assay needs it: off, once before the read, or before every kinetic cycle. Default duration is 3 seconds, adjustable to 999. Shaking matters for ELISAs and other solid-phase reactions and does very little for homogeneous liquid assays.
7. Set the incubator temperature if required, and let it equilibrate before you load.
8. Decide on PathCheck now — see 2.5. You cannot apply it after the read.
9. Click OK and save.

2.3 Lay out the plate template

The template tells the software which wells are samples, blanks, standards and controls. Without it you get a grid of raw numbers and have to do the bookkeeping yourself in Excel.

1. With the Plate section selected, open the Template Editor from the Home tab.
2. Select the wells that hold your blanks and assign them as Blank.
3. Select standards, assign them to a group, and enter their concentrations. The software builds the standard curve from these.
4. Select unknowns and assign them to a sample group. Replicates given the same name are averaged automatically, with SD and CV reported.
5. Close the Template Editor. Group sections appear in the Navigation Tree, one per group you defined.

Data reduction

Open the Data Reduction dialog to control what the reduced numbers actually are:

• Tick Use Plate Blank to subtract the average of the blank wells from every sample.
• For dual-wavelength reads, set the reduction formula to subtract the reference wavelength, e.g. !Lm1 − !Lm2 for a 450/620 nm ELISA.
• Choose the curve fit for standards — linear, 4-parameter logistic and about twenty others are available.

2.4 Load the plate and read

1. Open the drawer with the button on the top front right of the instrument, or Open/Close on the Home tab.
2. Seat the plate flat in the drawer with well A1 at the front right, against the moulded corner marker. The pusher holds the plate in place once the drawer closes.
3. Close the drawer.
   • If the incubator is on, the drawer closes itself after about 10 seconds to hold temperature.
4. Select the Plate section in the Navigation Tree and click Read.
5. Watch the first few wells appear. If everything reads at the rail, stop and check the plate material against your wavelength.
6. For kinetic runs: Interrupt pauses and lets you resume with Append. Stop ends the run permanently — you cannot restart or append after a Stop.
7. Save the document.

2.5 PathCheck — when to use it

In a cuvette the pathlength is fixed at 1 cm. In a plate the light goes down through the liquid, so pathlength depends on volume, and 200 µL of the same solution reads differently from 150 µL. PathCheck measures the actual depth of each well from the near-infrared absorbance of water and rescales every well to its 1 cm equivalent.

What this buys you: extinction-coefficient calculations directly from plate data, no standard curve needed, and tolerance for imprecise pipetting. Accuracy is within about ±4% of a real 1 cm cuvette reading.

Use PathCheck when

• Samples are aqueous, clear, and below 0.5 M salt.
• You want absolute concentrations from an extinction coefficient — NADH assays, A260 quantitation.
• Well volumes vary between wells.

Do not use PathCheck when

• The sample is turbid. Turbidity raises the 900 nm reading more than the 1000 nm reading and produces a falsely short pathlength. Cuvette Reference does not rescue this.
• The sample is strongly red or purple — Lowry, molybdate assays, haemoglobin, porphyrins. Their absorbance tails into the near-infrared.
• You're running an ELISA read against a standard curve, where it adds nothing.

Removing the plate's own absorbance

Raw plate OD includes the plastic itself, which doesn't scale with pathlength and must be removed before normalising. Two ways:

• Plate blank — if all wells hold the same volume, designate several wells as blanks, fill with buffer, and tick Use Plate Blank.
• Plate Background OD — if volumes differ, fill a clean plate with water, read it at your assay wavelengths, and enter the average as the background constant. Use water, not a dry plate.

3 · Getting data into Excel

This works identically for Cuvette Set and Plate sections.

3.1 Manual export

1. Open the data document containing the sections you want.
2. Click the Application menu (top-left icon), then Export › Export to XML XLS TXT.
3. In the Export dialog, tick the sections to include. All Plates, All Groups and All Notes select everything of that kind at once; otherwise tick individual sections.
   • Include the Group sections. They carry your sample names, means, SDs and calculated concentrations — the plate data alone is just a grid of numbers.
4. Choose the Plate Data Option: Raw, Reduced, or Both. Both is usually the right answer — you keep the untouched numbers alongside the processed ones.
5. Choose the Output Format:
   • Plate — a text matrix shaped like the physical plate. Good for eyeballing, awkward to analyse.
   • Columns — one column of values per well. This is the format you want for anything you intend to process further, pivot, or plot.
6. Click OK, navigate to your destination folder, enter a file name.
7. In Save As Type, choose .xls (list) or .xls (plate). Click Save.
8. Open the file in Excel. Excel may warn that the format doesn't match the extension — the file is a tab-delimited text file with an .xls extension, so this is expected. Click through it, then immediately re-save as .xlsx to get a real workbook.

3.2 Automatic export after every read

Worth setting up if you run the same assay repeatedly, or if you want a network-backed copy without thinking about it.

1. Open the protocol or data document.
2. Application menu › Auto Export.
3. Tick Save Data After Read, then click Add.
4. Set the destination: the protocol's own folder, or a folder you browse to.
5. Set File Format to .xls (list) or .xls (plate).
6. Choose Raw, Reduced, or Both.
7. Select Create a New File and tick Append Date to File Name and Append Time to File Name. Runs on the same day are then numbered sequentially rather than silently overwriting each other.
8. Click OK. Every read from now on writes a spreadsheet automatically.

3.3 Other output routes

• PDF report — Application menu › Print › Save As PDF, or select Molecular Devices (PDF Writer) in the Print dialog. Use Print Selected to pick which sections appear.
• .txt (list) or .txt (plate) — same content as the .xls options, tab delimited. Better if you're feeding a script.
• .xml — for LIMS or downstream data systems.
• Copy and paste — for a quick look, select cells in a Plate or Group section and paste straight into Excel.

4 · Finishing up

1. Remove the plate or cuvette. Never leave samples in the chamber.
2. Turn the incubator off if you used it.
3. Save the document, and export before you close it.
4. Close SoftMax Pro.
5. Switch the instrument off at the rear power switch. Molecular Devices recommends powering off when not in use.
6. Wipe the cuvette chamber and plate carrier if anything spilled. Log the run in the instrument book.

Periodic maintenance

• Clean the outer surfaces with a cloth lightly dampened with water, or a mild soap solution. Never spray cleaner into any opening.
• An air calibration is available under Operations tab › Calibration if readings drift. The instrument also self-calibrates before every endpoint read and before the first read of a kinetic run.
• The xenon lamp is rated for about 1 billion flashes, roughly two years of normal operation.
• Book an annual preventive maintenance visit from a Molecular Devices service engineer.
• Validate optical performance with the SpectraTest ABS2 plate (part 0200-6191) and the Cuvette Absorbance Validation Set (part 0200-2420) if your lab needs documented performance.

5 · Troubleshooting

Molecular Devices support: +1 408-747-1700, or Europe +44 (0) 118 944 8000. Have the serial number ABP01107 and your software version ready. The knowledge base is at support.moleculardevices.com.

Full PDF user guides are installed locally at C:\ProgramData\Molecular Devices\User Guides.

6 · Quick reference

Menu paths

File types