Table 4.

Troubleshooting

PROBLEM	STEP(S)	POSSIBLE REASON	SOLUTION
Poor cell recovery or high debris	6, 17, 31, 103 + 104–110	Low viability of starting cells	Improve cell culture and preparation.
		Incorrect spin speeds	Check spin speeds (note increased speed after fixation) in all centrifugations, particularly Step 51 onwards.
		Low starting cell number	Increase cell starting number.
		Harsh cell treatment	Limit vortexing of samples and handle carefully throughout the protocol, see Step 35 for correct procedure.
Poor mRNA staining	103 + 104–110	Oven temperature incorrect in Steps 76, 78, 85, 91 & 98.	Recalibrate oven.
		Oven temperature unstable in Steps 76, 78, 85, 91 & 98.	Carefully monitor oven temperature throughout protocol
		Incorrect amount of mRNA Target Probes used in Step 68.	Use Target Probes at 1:20
		Incorrect amount of Label Probes used in Step 96.	Use Label Probes at 1:100
		High residual volume in tubes, following washes in Steps 73, 81, 89 & 95.	Maintain residual volume at 100 µl.
		mRNA degradation in Steps 1–64. See Step 27 for example.	Ensure cells are in the growth phase before use. Work at 4 °C before fixation.
		Poor cell permeabilization and/or fixation in Steps 48, 53 & 65.	Check Reagent Setup (see Table 1). Use buffers prepared on the same day. Check incubation times and temperatures.
		Poor washing in Steps 71–73, 79–81, 87–89, 93–95, 99–101	Ensure all wash steps are followed, using the appropriate buffer at the required temperature.
High background in mRNA channel/high MFI of mRNA negative population	103 +104–110	Low viability/number of starting cells in Steps 1–24 and 36–40. Check viability in Step 31.	Improve cell culture and preparation. Use Viability stain to exclude dead cells.
		Incorrect amount of mRNA Target Probes used in Step 68.	Use Target Probes at 1:20
		Incorrect amount of Target Probes used in Step 96.	Use at 1:100
		Unclean flow machine/samples run too fast.	Clean the flow cytometer well before use. Run samples at ~2,000 events/second.
		Low residual volume following washes in Steps 73, 81, 89 & 95.	Residual volume should be 100 µl.
		Incorrect fixation time in Steps 50 + 76.	Check fixation time – longer fixes can increase background.
		Poor washing in Steps 71–73, 79–81, 87–89, 93–95, 99–101.	Ensure all wash steps are followed, using the appropriate buffer at the required temperature.
No HIVRNA+/Gag+cells detected	103 + 104–110	Low starting cell number in Step 23.	See “Limitations” for discussion of starting cell number.
		Poor mRNA staining.	Run a positive control sample to rule out assay issues.
Poor antibody staining	103 + 104–110	Clone unstable during protocol.	Test new antibody clones. See Box 4 for further information.
		Fluorochrome unstable during protocol.	Test additional fluorochromes. See Box 4 for further information.
		Incorrect incubation times/temperatures in Steps 43 & 61.	For anti-Gag KC57, stain for 1 hr (30 min at RT, 30 min at 4 °C)
		Non-optimal antibody concentration in Step 43.	Titrate all antibodies before use.
		Incorrect compensation.	See Box 4 for details
		Interactions between BV, BUV and BB polymer dyes.	Prepare antibody mixes with BD Horizon Brilliant Stain Buffer (Cat #563794) and compare to standard mix to determine if this buffer is required.