• Check the viability and passage number of the RAW cells. Higher passage numbers can lead to reduced phagocytic ability. Make sure the cells are healthy and not overgrown.
• Verify the IgG opsonization protocol and IgG concentration used. Suboptimal opsonization can reduce bead uptake. Try varying the IgG concentration to optimize opsonization.
• Use an IgG isotype control to confirm opsonization. Check bead uptake with non-opsonized beads as a negative control.
• Increase the bead to cell ratio and extend the incubation time. The kinetics of phagocytosis can vary, so allowing more time may increase bead internalization.
• Make sure beads and cells are incubated together with constant gentle agitation to maximize contact. Static incubation can reduce phagocytosis.
• Check the Fc receptor expression levels on the RAW cells. Low FcR expression can inhibit IgG-mediated phagocytosis.
• Monitor cell health during incubation. Toxicity or cell death during incubation could explain low bead uptake.
• Ensure assays are performed at 37°C as phagocytosis is an active process. Cooling cells can dramatically reduce phagocytic ability.

Hi Salah, Thanks for the detailed answer,

Some of the Raw cells have recently been received from ATCC and definitely, they are low passage, and they look healthy and okay, when I count them cell counter, the viability is 75%.

Surprisingly before receiving the new batch, the old batch was eating very well and then suddenly stopped and the new batch arrived, but still, they are not eating, maybe we have to figure out the opsonization protocol again and change IgG stock.

Thanks a lot. Appreciate.

Hello,

I'm sorry to hear you've been having trouble getting consistent results with your phagocytosis assays using RAW 264.7 cells. That can be really frustrating when cells stop behaving as expected.

A few thoughts on troubleshooting:

The new cells may need more time to recover after thawing and to regain optimal functionality. Even though they look healthy, some subtle functional differences may remain right after reviving frozen cells. Giving them more time to proliferate and stabilize in culture before assaying phagocytosis may help.

There could be an issue with the opsonization protocol or IgG stock that is resulting in less efficient opsonization and therefore decreased phagocytosis. As you mentioned, trying fresh IgG along with optimizing the opsonization steps may improve bead uptake.

Assay conditions like temperature, serum levels, or cell density may need to be re-optimized for the new batch of cells. Previous conditions may no longer be ideal.

if you can, check Fc receptor levels and compare them to previous effective batches could also provide clues.

Furthermore, A few things to consider about the beads:

Viability/integrity - Make sure the bead stock is not degraded or damaged. Look at them under a microscope to check for intact shape and structure. Damaged beads may not opsonize or be recognized properly.

Binding capacity - If the beads are older or degraded, their capacity to bind IgG could be reduced, resulting in lower opsonization levels. Testing binding with a saturating level of IgG can check for adequate coating.

IgG density - Related to binding capacity, you want to make sure the beads are saturated with IgG to promote efficient phagocytosis. Using higher IgG concentrations during opsonization can help maximize coating.

Non-specific binding - Some bead batches can have sticky surfaces that lead to IgG detaching or non-specific proteins attaching. This could interfere with opsonization and cell recognition.

Batch differences - Like with cells, different batches/lots of beads could have variability that affects performance. Testing a new batch or lot may help troubleshoot issues.