1. bookVolumen 69 (2022): Edición s1 (July 2022)
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Revista
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2453-6725
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25 Nov 2011
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2 veces al año
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Optimisation of flow cytometric detection of intracellular SLAMF receptor-associated adaptor proteins SAP and EAT-2

Publicado en línea: 18 Jun 2022
Volumen & Edición: Volumen 69 (2022) - Edición s1 (July 2022)
Páginas: 87 - 89
Recibido: 21 Apr 2022
Aceptado: 10 May 2022
Detalles de la revista
License
Formato
Revista
eISSN
2453-6725
Primera edición
25 Nov 2011
Calendario de la edición
2 veces al año
Idiomas
Inglés
INTRODUCTION

The SLAM (Signalling Lymphocyte Activation Molecule) family is a group of nine surface receptors (SLAMF1-9) expressed mainly on hematopoietic cells, that regulate the activation and differentiation of a wide range of immune cells involved in both innate and adaptive immune responses (Calpe et al., 2008; Cannons et al., 2011; Detre et al., 2012). SLAMF receptors interact with intracellular SLAM-associated protein (SAP)-related molecules that link SLAMF receptors to downstream intracellular signalling pathways. In T, NK, and NKT cells, SLAMF receptors interact with SAP. In mature B cells, SLAMF receptors induce a downstream cascade through EAT-2 (Tangye et al., 2002; Veillette, 2006; Radomir et al., 2017). This work’s aim is the optimisation of flow cytometry method for the detection of intracellular SAP and EAT-2.

MATERIAL AND METHODS

Peripheral blood was obtained from a healthy donor (M.S., corresponding author). The expression of intracellular/cytoplasmic SAP (cySAP) and EAT-2 (cyEAT-2) was studied with multiparametric flow cytometry. Samples were stained with combinations of monoclonal antibodies as shown in Table 1. Rat anti-SAP-PE monoclonal antibody (Invitrogen, USA) and rabbit anti-EAT-2 antibody (Proteintech, UK) with secondary donkey anti-rabbit IgG-PE antibody (BioLegend, USA) were used for the detection of cySAP and cyEAT-2, respectively. Four different commercial kits were employed for cell fixation and permeabilization: Fix & Perm Cell Permeabilization Reagents (Invitrogen, USA), Transcription Factor Buffer Set (BD Pharmingen, USA), IntraPrep Permeabilization Reagent and PerFix EXPOSE Kit (both Beckman Coulter, USA). Manufacturers’ instructions were closely followed. Samples were acquired using Navios EX flow cytometer (Beckman Coulter, USA). Data were analysed using Infinicyt software (Cytognos, Spain). The intensity of cySAP and cyEAT-2 expression was expressed as MFI (mean fluorescence intensity) value.

Panel of antibodies.

Antibodies and fluorochromes
Tube No. PB KO FITC PE ECD PC5.5 PC7 APC APC-A700 APC-A750
1 CD4 + CD20 CD45 - cyCTRL - CD5 CD10 CD3 CD19 CD8
2 CD4 + CD20 CD45 - cySAP - CD5 CD10 CD3 CD19 CD8
3 CD4 + CD20 CD45 - cyCTRL +sec.Ab - CD5 CD10 CD3 CD19 CD8
4 CD4 + CD20 CD45 - cyEAT-2 +sec.Ab - CD5 CD10 CD3 CD19 CD8
RESULTS AND DISCUSSION

Flow cytometric detection of intracellular (cytoplasmic or nuclear) versus cell surface antigens is significantly more laborious and time-consuming. It requires cell fixation, usually with paraformaldehyde, followed by permeabilization with Triton-X or saponin. Unfortunately, these procedures lead to significant cell damage, changes in forward and side scatter characteristics of cells and increased autofluorescence, which may complicate subsequent data analysis. Here, we compared four different commercial fixation and permeabilization kits with the aim to optimise methodology for the detection of intracellular/cytoplasmic SAP and EAT-2 proteins by flow cytometry.

In clinical flow cytometry laboratories, Fix & Perm and IntraPrep reagents belong to the most widely used kits for the detection of diagnostically important intracellular antigens. In our study, Fix & Perm showed unsatisfactory performance when used for cySAP staining as reflected by the very low MFI of stained cells (Figure 1, Table 2). For that reason, it was not further evaluated for cyEAT-2 detection. In contrast, the usage of IntraPrep resulted in a significantly stronger cySAP and cyEAT-2 staining in terms of MFI and a good signal-to-noise ratio. TFBS reagent led to a stronger cySAP staining than Fix & Perm but weaker than IntraPrep. Since the staining procedure using TFBS versus other reagents is significantly more time-consuming (approximately 3 hours versus approximately 1 hour), we decided not to evaluate it further. Finally, we used the PerFix kit. Originally developed for the detection of phosphorylated intracellular antigens, PerFix gave the best results in terms of cySAP and cyEAT-2 staining intensity and signal-to-noise ratio. Furthermore, it enabled separation of T cells, B cells, and NK cells based on the intensity of cySAP and cyEAT-2 expression. Previously, other authors who analysed cySAP and/or cyEAT-2 by flow cytometry reported only on the use of the traditional paraformaldehyde/Triton-X method (Schlums et al., 2015; Radomir et al., 2017; Gyurova et al., 2019). To conclude, we identified the staining procedure employing PerFix EXPOSE kit for cell fixation and permeabilization as the optimal method for cySAP and cyEAT-2 detection.

Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).

Flow cytometric analysis of SAP and EAT-2 adaptor proteins in lymphocytes.

MFI control (unstained) cells MFI stained cells
Mean SD CV Mean SD CV
cySAP T cells Fix&Perm 3.1 1.2 39.6 4.8 1.6 32.7
TFBS 2.7 1.3 47.9 17.1 11.5 67.5
IntraPrep 3.8 0.9 22.6 23.9 10.3 43.1
PerFix 4.9 2.0 41.3 73.9 54.2 73.3
B cells Fix&Perm 1.6 0.6 36.9 4.0 1.9 47.3
TFBS 1.1 0.2 15.2 10.0 7.0 70.3
IntraPrep 3.5 1.0 26.8 15.7 12.2 77.9
PerFix 2.7 0.7 27.1 28.9 27.3 94.8
NK cells Fix&Perm 1.8 0.6 30.9 4.5 2.4 54.0
TFBS 1.2 0.4 32.3 18.9 9.1 48.1
IntraPrep 4.0 0.8 19.5 22.6 9.5 42.1
PerFix 2.4 0.6 23.4 49.9 22.6 45.4
cyEAT-2 T cells Fix&Perm ND ND ND ND ND ND
TFBS ND ND ND ND ND ND
IntraPrep 4.0 0.9 21.7 83.9 46.3 55.1
PerFix 5.8 2.1 36.8 192.5 64.6 33.6
B cells Fix&Perm ND ND ND ND ND ND
TFBS ND ND ND ND ND ND
IntraPrep 3.7 0.9 24.2 63.1 37.3 59.1
PerFix 3.7 1.3 35.2 189.0 60.9 32.2
NK cells Fix&Perm ND ND ND ND ND ND
TFBS ND ND ND ND ND ND
IntraPrep 4.2 1.0 24.0 211.2 178.6 84.6
PerFix 3.1 0.7 23.1 537.7 227.3 42.3

Figure 1

Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).
Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).

Figure 1

Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).
Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).

Figure 1

Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).
Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).

Figure 1

Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).
Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).

Figure 1

Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).
Illustrative bivariate dotplots showing the results of cySAP (A) and cyEAT-2 (B) analysis by flow cytometry: control (unstained) cells (grey), T cells (light green), B cells (dark green), NK cells (blue), monocytes (yellow), neutrophils (violet) and eosinophils (pink).

Flow cytometric analysis of SAP and EAT-2 adaptor proteins in lymphocytes.

MFI control (unstained) cells MFI stained cells
Mean SD CV Mean SD CV
cySAP T cells Fix&Perm 3.1 1.2 39.6 4.8 1.6 32.7
TFBS 2.7 1.3 47.9 17.1 11.5 67.5
IntraPrep 3.8 0.9 22.6 23.9 10.3 43.1
PerFix 4.9 2.0 41.3 73.9 54.2 73.3
B cells Fix&Perm 1.6 0.6 36.9 4.0 1.9 47.3
TFBS 1.1 0.2 15.2 10.0 7.0 70.3
IntraPrep 3.5 1.0 26.8 15.7 12.2 77.9
PerFix 2.7 0.7 27.1 28.9 27.3 94.8
NK cells Fix&Perm 1.8 0.6 30.9 4.5 2.4 54.0
TFBS 1.2 0.4 32.3 18.9 9.1 48.1
IntraPrep 4.0 0.8 19.5 22.6 9.5 42.1
PerFix 2.4 0.6 23.4 49.9 22.6 45.4
cyEAT-2 T cells Fix&Perm ND ND ND ND ND ND
TFBS ND ND ND ND ND ND
IntraPrep 4.0 0.9 21.7 83.9 46.3 55.1
PerFix 5.8 2.1 36.8 192.5 64.6 33.6
B cells Fix&Perm ND ND ND ND ND ND
TFBS ND ND ND ND ND ND
IntraPrep 3.7 0.9 24.2 63.1 37.3 59.1
PerFix 3.7 1.3 35.2 189.0 60.9 32.2
NK cells Fix&Perm ND ND ND ND ND ND
TFBS ND ND ND ND ND ND
IntraPrep 4.2 1.0 24.0 211.2 178.6 84.6
PerFix 3.1 0.7 23.1 537.7 227.3 42.3

Panel of antibodies.

Antibodies and fluorochromes
Tube No. PB KO FITC PE ECD PC5.5 PC7 APC APC-A700 APC-A750
1 CD4 + CD20 CD45 - cyCTRL - CD5 CD10 CD3 CD19 CD8
2 CD4 + CD20 CD45 - cySAP - CD5 CD10 CD3 CD19 CD8
3 CD4 + CD20 CD45 - cyCTRL +sec.Ab - CD5 CD10 CD3 CD19 CD8
4 CD4 + CD20 CD45 - cyEAT-2 +sec.Ab - CD5 CD10 CD3 CD19 CD8

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