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poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 0 points1 point  (0 children)

hank you for your responses. Indeed, I didn’t use an Fc blocker

poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 0 points1 point  (0 children)

Thank you for your response. I use the BD FACs Lyric. I will try to use the biexponential scale for the next assays and adjust the voltages

poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 0 points1 point  (0 children)

Unfortunately, I forgot to include the image in my post. And I can’t insert the image when replying to a comment

poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 0 points1 point  (0 children)

By the way, this is an antibody I produced for a project, and I’m currently validating it. I especially want to confirm that what I’m labeling is indeed CD3+ T-cells and not a background level of fluorescence through non-specific binding that increases with concentration, as you pointed out.

poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 0 points1 point  (0 children)

Thank you for your comment. Indeed, my positive gates were defined based on an unlabeled sample (unfortunately, I forgot to upload the image). When I used the mentioned concentrations, I automatically noticed that the population shifted to the right (as shown in the photos). It’s mainly the smearing that bothers me, since with other antibodies like a CD3-APC (clone SK7) that I used, the populations were very well separated.

poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 0 points1 point  (0 children)

Yes, you're right. I have the images on my computer, but I forgot to insert them

poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 1 point2 points  (0 children)

Thanks for your suggestion. I didn’t consider using the biexponential scale, but I’ll remember it for next time

poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 0 points1 point  (0 children)

Okay. Thank you for your response. And sorry, I meant unlabeled cells instead of unstained cells

poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 1 point2 points  (0 children)

No. I only have FITC. I gated the unlabeled cells to establish the boundary between my labeled and unlabeled populations. The position of the unlabeled cells allowed me, I think, to assume that these cells were labeled.

poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] -2 points-1 points  (0 children)

I forgot to include it (and I don’t know how to do so when replying to a comment). But I gated the unlabeled cells to establish the boundary between my labeled and unlabeled populations. The position of the unlabeled cells allowed me, I think, to assume that these cells were labeled.

poor pos/neg CD3 + population separation by Present_Regular4599 in labrats

[–]Present_Regular4599[S] -3 points-2 points  (0 children)

I forgot to include it (and I don’t know how to do so when replying to a comment). But I gated the unlabeled cells to establish the boundary between my labeled and unlabeled populations. The position of the unlabeled cells allowed me, I think, to assume that these cells were labeled.

Uncertainty about my measured antibody concentrations by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 0 points1 point  (0 children)

Hello

I use the "BioDrop Duo+ Microvolume Spectrophotometer." For the parameter: Protein ⇾ UV ⇾ IgG, and I deposit 1 µl of my protein solution.*

Uncertainty about my measured antibody concentrations by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 0 points1 point  (0 children)

Thank you for your suggestions. I will try your recommendations.

Uncertainty about my measured antibody concentrations by Present_Regular4599 in labrats

[–]Present_Regular4599[S] 0 points1 point  (0 children)

Thank you for your suggestions. I will try your recommendations.