Primeflow

Manufactured by Thermo Fisher Scientific

PrimeFlow is a multicolor flow cytometry reagent system designed for the detection and analysis of multiple cellular biomarkers simultaneously. It enables the quantification of gene expression at the single-cell level through the simultaneous measurement of multiple cellular proteins and RNA targets.

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PrimeFlow RNA assay PrimeFlow RNA PrimeFlow assay kit PrimeFlow RNA Assay Kit Primeflow assay

5 protocols using primeflow

Cytokine and Signaling Pathway Analysis by Flow Cytometry

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Flow cytometry experiments were performed using a FACS Celesta (BD Biosciences) and analyzed using FlowJo software (v.10.3). All samples from all experiments are gated on viable cells utilizing Fixable Viability Dye eFluor 780 (65-0865-14, eBioscience). Intracellular staining for cytokines, pCAD, and pS6 was performed with BD Fixation/Permeabilization solution (BD Biosciences, 554714). For PRIME Flow® studies, samples were prepared in accordance with the provided protocol (ThermoFisher). Data are reported as mean fluorescence intensity and displayed as a percentage of events recorded relative to the mode values for each data set collected.

Claiborne M.D., Sengupta S., Zhao L., Arwood M.L., Sun I.M., Wen J., Thompson E.A., Mitchell-Flack M., Laiho M, & Powell J.D. (2022). Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge. Science immunology, 7(71), eabh4271.

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Multiparametric Flow Cytometry of Skin Immune Cells

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Single cell suspensions obtained from full thickness samples or after stimulation with S. epidermidis and MRSA were first labeled with live/dead detection kit (Yellow Amine, Thermo Fisher Scientific) and then with the following fluorescently labeled antibodies: CD45-Alexa Fluor 700, TCR GD-PE-Cy7, CD31-PacBlue, CD104-FITC, CD325-PerCPCy5.5, and CCRL1-PE (Biolegend, San Diego, CA, United States). We also stained cells with fluorescently labeled antibodies for TLR1-BV570, TLR2-PE, TLR6-BV605, and TCR GD 1 FITC (Biolegend, San Diego, CA, United States). P-2 mRNA was detected using an amplified signal FISH technique (PrimeFlow; Affymetrix/eBioscience-Thermo Fisher Scientific). For mRNA detection, target probe hybridization was performed using type 1 (AlexaFluor647) probes for P-2 as described (43 (link)). Approximately 20,000 cell events were acquired from each sample on flow cytometer equipped with 405 nm, 488 nm, 642 nm, and 785 nm (SSC) lasers (Fortessa X-50, BD Immunocytometry Systems, San Jose, CA, United States). Spectral compensation was completed using single color control samples and antibody capture beads (BD Biosciences). Data were analyzed using FlowJo version 10.2 (TreeStar).

Pastar I., O’Neill K., Padula L., Head C.R., Burgess J.L., Chen V., Garcia D., Stojadinovic O., Hower S., Plano G.V., Thaller S.R., Tomic-Canic M, & Strbo N. (2020). Staphylococcus epidermidis Boosts Innate Immune Response by Activation of Gamma Delta T Cells and Induction of Perforin-2 in Human Skin. Frontiers in Immunology, 11, 550946.

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Multiparameter Flow Cytometry of Malaria Transcripts

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RNA FISH was carried out using PrimeFlow (ThermoFisher) according to the manufacturer’s instructions and stained for DNA with Hoechst33342. Custom probes were designed and synthesized against unique regions of the AP2-G (AlexaFluor 488), CDPK5 (AlexaFluor 647), LSD2 (AlexaFluor 568) and AP2 PF3D7_1139300 (AlexaFluor 568) transcripts by the manufacturer (Supplementary Table S5). Detection was carried out using a BD LSR II flow-cytometer using 350nm, 488nm, 561nm, and 650nm lasers, respectively. To ensure analysis of individual parasites, a single uniform population of parasites (RBC membranes are removed during fixation/permeabilization) was triple gated on FSC-A vs SSC-A, FSC-H vs FSC –A and SSC-H vs SSC-A. Fluorescence gates were set based on fluorescence-minus-one controls (see Extended Data Fig. 9). Mature schizonts, as identified by maximal DNA content and high CDPK5 expression, were analyzed for expression of AP2-G and either LSD2 or PF3D7_1139300.

Poran A., Nötzel C., Aly O., Mencia-Trinchant N., Harris C.T., Guzman M.L., Hassane D.C., Elemento O, & Kafsack B.F. (2017). Single-cell RNA-seq reveals a signature of sexual commitment in malaria parasites. Nature, 551(7678), 95-99.

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TCDD-induced FasL and Cyp1a1 expression

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Splenocytes were treated with VH (0.1% DMSO) or TCDD (30 nM) on day 1 and incubated overnight then the PrimeFlow was initated according to the manufacturer’s protocol (Thermo Fischer Scientific) on day 2. Briefly, the cells were stained with CD19-PE/Cy7 and FasL-PE in FCM buffer containing 0.01% sodium azide and Fc block for 20 min at RT in the dark. Cells were then fixed overnight using the fixation buffers provided in the kit. On day 3, the cells were incubated with target probes Type 1-Bactin (APC) and Type 4-Cyp1a1 (FITC) at 40°C. Finally on day 4, the cells were incubated with amplification and hybridization buffers and signal was detected using fluorescent label probes. Cells were analyzed using an ACEA Novocyte and guidance for gate settings were made with FMO controls. Data analysis was done with NovoExpress software.

Kummari E., Rushing E., Nicaise A., McDonald A, & Kaplan B.L. (2020). TCDD Attenuates EAE Through Induction of FasL on B Cells and Inhibition of IgG Production. Toxicology, 448, 152646.

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Multiparametric Immune Cell Profiling

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Isolated cells were washed in FACS staining buffer and incubated with anti-CD16/32 (clone 2.4G2) to block FcRs for 10 mins followed by an incubation with Live/Dead fixable yellow dead cell stain (ThermoFisher) and fluorescent-conjugated monoclonal antibodies (mAbs) for 30 min.
The following anti-mouse mAbs were used for analysis: CD45 (30-F11; BioLegend), CD3 (17A2; BioLegend), F4/80 (BM8; BioLegend), CD11b (M1/70; BioLegend), Ly6C (HK1.4; BioLegend), Ly6G (1A8; BioLegend), CD335 (29A1.4; BioLegend), MHC II (M5/114.15.2; BD Biosciences), CD206 (C068C2; BioLegend). Depending on the experiment, samples were either immediately analyzed by flow cytometry using a Sony SP6800 Spectral Analyzer or further processed to determine Perforin-2 mRNA levels. Branched oligonucleotide signal amplification was used to determine Perforin-2 mRNA levels in individual cells (PrimeFlow; ThermoFisher Scientific).
Briefly, single cell suspensions were stained for surface antigens as described above, then fixed, permeabilized, and incubated with probes specific for Perforin-2 transcripts (Assay Id: VB1-20172-PF; ThermoFisher Scientific). Cells were then subjected to a series of signal amplification cycles and then analyzed by flow cytometry as described above using FlowJo software (BD Biosciences).

Gayle P., McGaughey V., Hernandez R., Wylie M., Colletti R.C., Nguyen K.L., Arons M., Padula L., Štrbo N., & Schesser K. (2020). Perforin-2 limits pathogen proliferation at the maternal-fetal interface.

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