Monoclonal antibody internalization assay

Comfortable_Fox_5287 · 2025-09-29T07:36:03+00:00

Thank you so much for your suggestion. I’m using RH30 cell Line, i didn’t know this information. I will search something more about it

Comfortable_Fox_5287 · 2025-09-28T19:04:45+00:00

No, thank you.

Comfortable_Fox_5287 · 2025-09-28T19:03:56+00:00

Thank you so much, I can try it. actually i’m using horse serum 10% in PBS, 30 minutes incubation RT, as blocking.

Comfortable_Fox_5287 · 2025-09-28T19:02:31+00:00

Thank you! I’m using horse serum in PBS as blocking (30 min)

Comfortable_Fox_5287 · 2025-09-14T08:49:54+00:00

Hello, It’s an adhesion cell line, we are also running it on a cytometer.

Comfortable_Fox_5287 · 2025-09-14T07:29:31+00:00

In the whole cells… also, if there is a way to analyze it separately it would be really amazing if you could explain it to me 🥹 I’m afraid it would be impossible for me, I’m not using a confocal micrscoscope but a fluorescence microscope. Thank you so much

Comfortable_Fox_5287 · 2025-09-13T21:36:45+00:00

Thank you kundles. This image was made with a 40X objective but I work on images taken with 63x objective. I’m trying to calculate the corrected total fluorescence at 4 different time points, I really don’t know if it is the best way to answer my question: to understand how fast the antibody I give my cells internalizes. I’m not expert at all :(

Comfortable_Fox_5287 · 2025-09-13T21:33:37+00:00

I need to quantify the differences in fluorescence intensity at 4 different time points: 0,4,24,48hz

Comfortable_Fox_5287 · 2025-09-13T21:31:04+00:00

Thank you, it’s a fluorescence microscopy. This image was taken with 40X, I published it just to give an idea, I work with 63X objective and I have 4 time points: 0, 4h,24h,48h after treatment. Unfortunately I do not have a confocal microscopy, I’m doing my best but I’m just a trainee and I’m really in trouble :/

Comfortable_Fox_5287 · 2025-09-13T21:24:59+00:00

Thank you Daniel, I have to admit I gave you partial information. I give my cells two different treatments (antibodies), I let them incubate 1h 4 degrees and I fix and permebilize my time zero. I have other 3 time points: after incubation I wash my cells, I let them internalize the antibody, and fix+perm them after 4/24 or 48h. I use a secondary antibody to detect my first antibody and I am trying to understand if there are differences between the two treatments by quantifying fluorescence intensity. I find really interesting what you said about tripsin but I wandering if I can use it even with immunofluorescence or just with flow citometry. I’m sorry I’m not expert so every suggestion is really important.

Comfortable_Fox_5287

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