Troubleshooting

Possible Cause:
If the kit-supplied Calibration Standards do not have % RET values within the ranges supplied by Litron, the CD71-FITC antibody is not working correctly.

Suggestion:
Prepare new Labeling Solutions. Stain the Calibration Standards and experimental samples with the new Labeling Solution and re-calibrate the flow cytometer.

Possible Cause:
You are taking samples from mature animals and % RET decreases with age.

Suggestion:
Ensure that you are comparing % RET values from age-matched animals.

Possible Cause:
The blood was fixed or stored in Fixative at temperatures warmer than -75 °C. This has caused a loss of the CD71 receptor and a corresponding loss of fluorescence.

Note: The chance of this happening is greatest in the first few days after blood samples have been fixed. Once transferred to Long Term Storage Solution (LTSS), samples can withstand wider temperature fluctuations. When in Fixative, the samples must be kept colder than –75 °C.

Suggestion:
Once this loss occurs, samples can no longer be reliably analyzed. To prevent recurrence:

Maintain your ultracold freezer at –75 °C or colder.
•
Use a chest (i.e., horizontal) freezer rather than an upright (i.e., vertical) freezer.
Because cold air sinks, chest freezers maintain temperature far better than upright freezers.
If you must use an upright (i.e., vertical) freezer, use the alternate fixing procedure.
•
Ensure Fixative and fixed blood samples are stored away from the freezer doors and are not exposed to room temperature. Even brief exposure to room temperature can compromise samples.
•
Transfer blood samples from Fixative to LTSS as described in the manual.

Possible Cause:
The most common cause of this extension is cellular aggregation. This aggregation can be caused by clotting during blood collection, or when the temperature of the fixed samples rises above –75 °C (see previous question).

Note: When faced with this problem, flow users often exclude these cell aggregates by simply making the "Single Cells" region smaller. However, this does not solve the problem because there is no way to gate out ALL aggregates. Those that remain will then appear as an extension of the NCE (lower left) population on the other plots.

Suggestion:
Once this occurs, samples can no longer be successfully analyzed. To prevent recurrence:

Ensure blood is free flowing during collection.
•
Keep Fixative and fixed blood samples at –75 °C or colder (see previous question).
•
Collect blood directly into:
(1) The kit-supplied Anticoagulant Solution (preferred).
or
(2) EDTA or another anticoagulant. After collection, transfer into the kit-supplied Anticoagulant Solution (as described in the manual).

Feel free to send us new questions or example plots to examine. We are always here to help!