I have recently coupled some magplex beads to protein, using the biorad kit and beads following a standard protocol from my supervisor that has worked well previously.
When I went to count these (used 1ul beads in 9ul diluent) there was barely anything in the square but I saw way more beads at the edge of the haemocytometer outside of the square? Per calculations from the square there were only about a million beads per ml instead of around 8-10 million/ml (the amount it should be) going by the counts. I doubted this so I ran a test on the luminex. I did a column with beads diluted as though there was 8 million/ml, one where I assumed they were all about 3mil/ml to avoid wasting too many beads. Both columns came up just fine when I read the plate so now I'm even more confused. Does anyone have any idea how to resolve this or a standard counting protocol as I don't have access to a cell counter.
Hi Sam Marcs, the issue you encountered with counting the magplex beads using a hemocytometer can be challenging without a dedicated cell counter. However, there are a few potential reasons for the discrepancy you observed:
- Bead distribution: Sometimes, when using a hemocytometer, the beads may not distribute evenly within the counting square. This can lead to an underestimation of the bead count. It's important to ensure thorough mixing and consistent pipetting during the dilution process to achieve a more representative distribution.
- Bead aggregation: Beads can sometimes aggregate or clump together, making them difficult to disperse uniformly. This can result in uneven distribution within the counting square, leading to inaccurate counts. Proper vortexing or sonication can help in dispersing the beads more effectively.
- Light scattering: The small size of the beads may cause them to scatter light, making it challenging to distinguish individual beads accurately using a hemocytometer. In such cases, using a dedicated cell counter or an alternative counting method, such as flow cytometry, can provide more reliable results.
Also, considering that your luminex test results were consistent with the expected concentrations, it suggests that the bead preparation and dilution were likely accurate. However, it's always a good practice to optimize your counting method and validate it against alternative techniques if possible.
If access to a cell counter is not available, you can explore other methods to estimate bead concentration, such as using a spectrophotometer to measure the absorbance at specific wavelengths or employing imaging software for automated counting based on images of diluted bead samples.
Cheers!
-H-
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