. Author manuscript; available in PMC: 2018 Oct 1.

Published in final edited form as: Nat Protoc. 2017 Sep 7;12(10):2029–2049. doi: 10.1038/nprot.2017.079

TABLE 1.

Example reagent preparation.

Reagent	Preparation	For 1 tube	For 12 tubes^*
*Viability stain*	1:500 in cold PBS (2 × stock)	100µl (99.8µl PBS + 0.2µl Viability stain)	1300µl (1297.4µl PBS + 2.6µl Viability stain)
*Surface stain* Use immediately	Anti-CD3 BV605, anti-CD4 PE-Cy7, anti-CD8, CD14 and CD19 BV510.	19µl (Anti-CD3 BV605 (5µl), anti-CD4 PE-Cy7 (5ul) anti-CD8, CD14 and CD19 BV510 (all 3µl)	247µl (Anti-CD3 BV605 (65µl), anti-CD4 PE-Cy7 (65ul) anti-CD8, CD14 and CD19 BV510 (all 39µl)
*Fixation I* Use immediately	1 part Fixation Buffer 1A for 1 part Fixation buffer 1B	1 ml (500 µl Fixation Buffer 1A + 500 µl 1B)	13 ml (6.5 ml Fixation Buffer 1A + 6.5 ml Fixation Buffer 1B)
*Permeabilization Buffer* Use immediately	1:10 Permeabilization Buffer, 1:1000 RNAsin I, 1:100 RNAsin II in H₂0	3 ml (300 µl Permeabilization buffer, 3 µl RNAsin I, 30 µl RNAsin II + 2.67 ml H₂0)	39 ml (3.9 ml Permeabilization buffer, 39 µl RNAsin I, 390 µl RNAsin II + 34.71 ml H₂0)
*Fixation II* Use immediately	1:8 Fixation Buffer 2 in Wash Buffer	1 ml (125 µl Fixation Buffer 2 + 875 µl Wash Buffer)	13 ml (1.625 ml Fixation Buffer 2 + 11.375 ml Wash Buffer)
*GagPol probes* Warm to 40 °C	1:20 Target mRNA probes in Target Probe Diluent	100 µl (5 µl Gag probe + 5µl Pol probe + 90 µl Target Probe Diluent.	1.3 ml (65 µl Gag probe + 65µl Pol probe + 1.170 ml Target Probe Diluent.
*Overnight storage buffer*	1:1000 RNAsin I in Wash Buffer	1 ml (1 µl RNAsin I + 999 µl wash buffer)	13 ml (13 µl RNAsin I + 12.987 ml wash buffer)
*Label probes* Warm to 40 °C	1:100 Label Probes in Label Probe Diluent	100 µl (1 µl + 99 µl)	1.3 ml (13 µl + 1.287 ml)

Sufficient reagent for one spare test is prepared to take into account pipetting error.