Table 4 ∣.

Troubleshooting table

Step	Problem	Possible reason	Solution
2	OP-Puro precipitates out of solution	Unbalanced reaction	Add HCl dropwise (1 μl) and mix until OP-Puro fully dissolves. Readjust pH to 6.4–6.6 by adding NaOH dropwise (1 μl)
47	Formation of a precipitate after washing azide–alkyne cycloaddition reaction with permeabilization buffer	The permeabilization buffer was not vacuum-filtered	FBS and saponin might contain solid particles that might cause the formation of a white precipitate after contact with CuSO4. After adding FBS and dissolving saponin in PBS, vacuum-filter the solution
51	Not enough events recorded	Cells were lysed because the permeabilization step was too long	Optimize the permeabilization time for each cell type to ensure cells are not lysed
		Incorrect cell counting was performed, and the wrong number of cells was taken for staining	Make sure your cell-counting technique is accurate
		Fluidic system in flow cytometer becomes clogged	Run water or a cleaning solution through the flow cytometer to eliminate any cells and/or debris that might be clogging the fluidic system
	No signal for some fluorophores	Fluorescence has been quenched during fixation, permeabilization or after Click-iT reaction	Certain fluorophores are damaged or quenched by the subsequent fixation and azide–alkyne cycloaddition reaction. This includes any PE or PE-conjugated dyes; these dyes should be strictly avoided. We have confirmed the stability of the following fluorophores through this reaction: FITC, PERCP-Cy5.5, APC, APC-eFluor780, Alexa Fluor 660, Alexa Fluor 700. Any alternative fluorophores should be tested before use
	No OP-Puro signal	Click-iT reaction did not take place	Make sure the cells are permeabilized so the AF555 and the click-iT reagents can penetrate the cell membrane in order to react with the OP-Puro to produce fluorescence
			Make sure you perform the Click-iT reaction at room temperature. The reaction time is optimized for this condition. If performed on ice, the time required for the azide–alkyne cycloaddition reaction might increase
		Inadequate i.p. injection and no OP-Puro incorporation	When performing the i.p. injection, avoid injecting into any organ, because this would prevent OP-Puro incorporation into the bone marrow
		Wrong channel selected in the flow cytometer	AF555 has an excitation maximum of 555 nm and an emission maximum of 580 nm. Therefore, a laser line of 488/532 should be used to detect fluorescence
		OP-Puro precipitated out of solution	Make sure the aliquot of OP-Puro has not precipitated out of solution before injecting the mice. Readjust the pH to dissolve any precipitate formed, driving the pH back to 6.4. If the formation of precipitate is reiterative, discard that aliquot and use a new one
	Low OP-Puro signal	AF555 became inactive because of light exposure	Store AF555 in the dark at −20 °C
		Wrong OP-Puro dose injected	Accurately weigh the animals and calculate the dose appropriately
		Incorrect flow cytometer setup	Make sure that the voltage is high enough to produce a visible signal