TITLE: Protocol for assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells-Human
AUTHORS: Marco A. de Oliveira, Lilian H. Florentino, Thais T. Sales, Rayane N. Lima, Luciana R. C. Barros, Cintia G. Limia, Mariana S. M. Almeida, Maria L. Robledo, Leila M. G. Barros, Eduardo O. Melo, Daniela M. Bittencourt, Stevens K. Rehen, Martín H. Bonamino, Elibio Rech

[DESCRIPTION]
This protocol describes the assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells in human.

[STEPS]
SECTION: PBMC isolation ● Timing 1.5 h
1. After performing the sterilization procedure in the safety cabinet, couple the syringe containing 20 mL PBS to the leukocyte filter, cut the filter tube extremity, and put it into a 50 mL tube.
 

SECTION: PBMC isolation ● Timing 1.5 h
2. Press the syringe to wash the leukocyte filter. Repeat this step if more cells are needed.
SECTION: PBMC isolation ● Timing 1.5 h
4. Centrifuge at 800 x g, 20 min, in a swinging-bucket rotor, without breaking.
 

SECTION: PBMC isolation ● Timing 1.5 h
5. With a 10 mL serological pipette, very carefully remove the PBMC “white ring” on top of the Ficoll-Paque and put it into a fresh tube.
 

SECTION: PBMC isolation ● Timing 1.5 h
6. To wash the cells, complete the volume to 50 mL with PBS and centrifuge at 400 x g, 5 min, 10 °C. Remove the supernatant.

SECTION: PBMC isolation ● Timing 1.5 h
7. Repeat the wash step twice.
SECTION: PBMC isolation ● Timing 1.5 h
8. Resuspend the cells in 5 mL-10 mL PBS and maintain them on ice.

SECTION: PBMC isolation ● Timing 1.5 h
9. Count the cells with a Neubauer chamber by adding Trypan blue solution at 4% for cell viability staining.
 

SECTION: PBMC electroporation ● Timing 1 h
10. Add 10x106 cells per electroporation reaction separating in individual microtubes.
SECTION: PBMC electroporation ● Timing 1 h
11. Centrifuge the tubes at 400 x g, 5 min, 10 °C.

SECTION: PBMC electroporation ● Timing 1 h
12. Remove all supernatant with a pipette and resuspend the cells in a total of 100 µL of 1SM electroporation buffer + plasmids (integrase + target) per reaction.

SECTION: PBMC electroporation ● Timing 1 h
13. Immediately insert the cells in the cuvette and electroporate using program U-014 on Nucleofector 2b.
 

SECTION: PBMC electroporation ● Timing 1 h
14. Very carefully, resuspend the cells in warm RPMI/FBS 20% (no antibiotics) and plate them in total 500 µL in a 12-well plate. After 16 hours, add 500 µL RPMI/FBS 10% + P/S antibiotics + 50 U/mL IL2 + L-Glutamine.
 



SECTION: Integrase activity evaluation in PBMCs at 2 h
15. 
 
Collect the cells and place them in a microtube.
SECTION: Integrase activity evaluation in PBMCs at 2 h
16. Centrifuge at 400 x g, 5 min. Remove the supernatant.

SECTION: Integrase activity evaluation in PBMCs at 2 h
17. Wash the cells with 1 mL of cold PBS. If DNA will be extracted, separate an aliquot at this time.

SECTION: Integrase activity evaluation in PBMCs at 2 h
18. Repeat the centrifugation 2X to wash the cells.
 

SECTION: Integrase activity evaluation in PBMCs at 2 h
19. Resuspend the cells in 300 µL of cold PBS and place them in the flow cytometer tub.
 

SECTION: Integrase activity evaluation in PBMCs at 2 h
20. Add 5 µL of 7-AAD and incubate at 4 °C for 20 min.
 


SECTION: Integrase activity evaluation in PBMCs at 2 h
21. Acquire at least 10,000 cells at the viable gate to evaluate eGFP expression. For a higher sensitivity, acquire more cells.
 

SECTION: HEK293T transfection with Calcium Phosphate ● Timing 1 h
22. Plate 4x106 HEK293T cells per well of a 75 cm2 plate at least 16 h before transfection in DMEM complete medium.
 

SECTION: HEK293T transfection with Calcium Phosphate ● Timing 1 h
23. Replace the medium before transfection, adding only 10 mL of DMEM complete medium.

SECTION: HEK293T transfection with Calcium Phosphate ● Timing 1 h
24. Mix the plasmids (integrase + targets, 5 µg each) to 500 µL of 2X CaCl2.
 

SECTION: HEK293T transfection with Calcium Phosphate ● Timing 1 h
25. Next, agitate CaCl2 using a vortex at full speed and add 500 µL of the HBS solution drop-by-drop very slowly. Make bubbles on the solution with a Pasteur pipette. Let it rest for an instant.
 


SECTION: HEK293T transfection with Calcium Phosphate ● Timing 1 h
26. Very carefully add drop-by-drop at the cells covering all the flask with circular movements avoiding two drops at the same spot. Mix the solution with ∞ movement and place the cells in the incubator.
SECTION: Integrase activity evaluation in HEK293T cells.● Timing 2h
27. Integrase activity was evaluated by flow cytometry 24 h after transfection. For optimization, several time points should be evaluated. In our hands, 72 h after electroporation, eGFP expression achieved its maximum (1-35% of positive cells).
SECTION: Cell expansion of the hES cell line-Passage cells
28. Culture hES cells in a 100-mm culture dish until the cells cover the dish area at 60-70% confluence. This occurs at approximately 5-6 days.
 

SECTION: Cell expansion of the hES cell line-Passage cells
29. Prior to starting the dissociation, coat the fresh 100-mm culture dishes with 6 mL/dish of cold Geltrex™ and place it into the incubator at 37 °C for at least 30 min.

SECTION: Cell expansion of the hES cell line-Passage cells
30. Remove Geltrex™, and immediately add 9 mL/dish of fresh growth mTeSR™ 1 medium to prevent drying.
SECTION: Cell expansion of the hES cell line-Passage cells
31. Place the fresh culture dishes into the incubator at 37 °C.

SECTION: Cell expansion of the hES cell line-Passage cells
32. Carefully aspirate the old growth medium from the cell culture dish.
SECTION: Cell expansion of the hES cell line-Passage cells
33. Wash the dish with 4 mL of DPBS without calcium and magnesium (DPBS-/-) and remove immediately.

SECTION: Cell expansion of the hES cell line-Passage cells
34. Add 4 mL of Accutase™ into the 100-mm culture dish, ensure that cover of dish area.

SECTION: Cell expansion of the hES cell line-Passage cells
35. Incubate the cell suspension at 37 °C for 5 min.
 



SECTION: Cell expansion of the hES cell line-Passage cells
36. Add 3 mL of DMEM/F12 and gently triturate the clusters across the dish surface.

SECTION: Cell expansion of the hES cell line-Passage cells
37. Scrape the attached cells using a cell scraper.
SECTION: Cell expansion of the hES cell line-Passage cells
38. Transfer the unattached cell suspension into a 15 mL conical tube containing 3 mL of DMEM/F12 using a 5 mL serological pipette.

SECTION: Cell expansion of the hES cell line-Passage cells
39. Spin down at 100 x g, 4 min, 25 °C.

SECTION: Cell expansion of the hES cell line-Passage cells
40. Aspirate and discard the supernatant.
SECTION: Cell expansion of the hES cell line-Passage cells
41. Resuspend the cell pellet with 5 mL of fresh growth mTeSR™ 1 Medium using a 5 mL serological pipette.

SECTION: Cell expansion of the hES cell line-Passage cells
42. Add the cell suspension 1 mL/dish into fresh 100-mm culture dishes prepared previously at step 28-31.
SECTION: Cell expansion of the hES cell line-Passage cells
43. Incubate the cells at 37 °C, 95% O2, 5% CO2 in humidified air.

SECTION: Cell expansion of the hES cell line-Passage cells
44. After 24 h, replace the old growth medium with fresh mTeSR™ 1 medium.
SECTION: Cell expansion of the hES cell line-Passage cells
45. Split the cells every 5-6 days.
SECTION: Maintenance of hES cell culture ● Timing 5 min daily
46. Carefully aspirate the old growth medium from the cell culture dish.
SECTION: Maintenance of hES cell culture ● Timing 5 min daily
47. Replace with 10 mL of fresh growth mTeSR™ 1 Medium.

SECTION: Maintenance of hES cell culture ● Timing 5 min daily
48. Incubate the cells at 37 °C, 95% O2, and 5% CO2 in humidified air until 60-70% confluent.

SECTION: Maintenance of hES cell culture ● Timing 5 min daily
49. Replace the medium daily.
 

SECTION: Cells preparation for electroporation ● Timing 6 d
50. Replace the old growth mTeSR™1 Medium for 6 days.
 

SECTION: hES cell electroporation ● Timing 1 h
51. (Optional) Prior to starting dissociation, add iROCK (10 micromolar (µM)) in the 100-mm cell culture dish for 60 min.

SECTION: hES cell electroporation ● Timing 1 h
52. Coat the fresh 12-well plate with 6 mL of cold Geltrex™ (0.5 µL/well) as described in steps 29-31.

SECTION: hES cell electroporation ● Timing 1 h
53. Aspirate Geltrex™ and add 0.5 µL/well of antibiotic-free fresh mTeSR™1 Medium with iROCK (10 micromolar (µM)).

SECTION: hES cell electroporation ● Timing 1 h
54. Carefully aspirate the old growth medium from the cell 100-mm culture dishes.
SECTION: hES cell electroporation ● Timing 1 h
55. Wash with 4 mL/dish of DPBS-/- and remove immediately.
SECTION: hES cell electroporation ● Timing 1 h
56. Add 4 mL/dish of Accutase™, ensure that cover of dish area.

SECTION: hES cell electroporation ● Timing 1 h
57. Incubate the cell suspension at 37 °C for 5-10 minutes.
 


SECTION: hES cell electroporation ● Timing 1 h
58. Add 3 mL/dish of DMEM/F12 and scrape the attached cells using a cell scraper.

SECTION: hES cell electroporation ● Timing 1 h
59. Gently triturate the clusters across dish surface.
SECTION: hES cell electroporation ● Timing 1 h
60. Transfer the unattached cell suspension using a 5 mL serological pipette into a 50 mL conical tube containing 10 mL of DMEM/F12.

SECTION: hES cell electroporation ● Timing 1 h
61. Spin down at 100 x g, 4 min, 25 °C.

SECTION: hES cell electroporation ● Timing 1 h
62. Remove the supernatant and wash with 10 mL of DPBS-/-.

SECTION: hES cell electroporation ● Timing 1 h
63. Count the cells with Trypan blue dye exclusion.
 

SECTION: hES cell electroporation ● Timing 1 h
64. Spin down at 100 x g, 4 min, 25 °C.

SECTION: hES cell electroporation ● Timing 1 h
65. Aspirate and discard the supernatant.
 

SECTION: hES cell electroporation ● Timing 1 h
66. Resuspend the cells with ice-cold DPBS-/-.
SECTION: hES cell electroporation ● Timing 1 h
67. Gently mix the suspension cells and transfer 2 mL/15 mL conical tube (1x106 cells per tube).
SECTION: hES cell electroporation ● Timing 1 h
68. Spin down at 100 x g, 4 min, 4 °C.

SECTION: hES cell electroporation ● Timing 1 h
69. Remove the supernatant and resuspend the pellet with 1SM electroporation buffer mix with the plasmids in a total volume of 100 µL.
 


SECTION: hES cell electroporation ● Timing 1 h
70. Immediately transfer the suspension cells into the 0.2 cm electroporation cuvette, preventing bubbles.
SECTION: hES cell electroporation ● Timing 1 h
71. Electroporate the cells by using the Nucleofector 2B program A-23.
 

SECTION: hES cell electroporation ● Timing 1 h
72. Immediately add 1 mL of fresh mTeSR™1 Medium with iROCK (10 micromolar (µM)) into the electroporation cuvette using a micropipette with a 1000 µL tip to avoid low cell viability.

SECTION: hES cell electroporation ● Timing 1 h
73. Immediately transfer the cell suspension into a 15 mL conical tube containing 0.5 mL of antibiotic-free fresh mTeSR™1 Medium with iROCK (10 micromolar (µM)).
 


SECTION: hES cell electroporation ● Timing 1 h
74. Gently mix the cell suspension and transfer into three wells (0.5 mL/well) of the coated Geltrex™ 12-well plate previously prepared in steps 51-52.
SECTION: hES cell electroporation ● Timing 1 h
75. Incubate the cells at 37 °C, 95% O2, 5% CO2 in humidified air.

SECTION: hES cell electroporation ● Timing 1 h
76. After 24 h, replace the old growth medium with antibiotic-free fresh mTeSR™1 medium.
SECTION: Postelectroporation ● Timing 2 d
77. After 48 h of transfection, evaluate the integrase activity by flow cytometry.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
78. Harvest cells using Accutase™ as described in step 24.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
79. Remove the old growth medium and wash with 0.5 mL/well of DPBS-/-, remove immediately.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
80. Add 0.4 mL/well of Accutase™.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
81. Incubate the cells in the incubator at 37 °C for 5-10 minutes.
 


SECTION: Preparation of sample and flow cytometry ● Timing 2 h
82. Add 0.5 mL/well of DMEM/F12 into a 15 mL conical tube containing 5 mL of DMEM/F12.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
83. Spin down at 100 x g, 4 min, 25 °C.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
84. Aspirate and discard the supernatant.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
85. Wash the cells with 2 mL of ice-cold DPBS-/-.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
86. Spin at 100 x g, 4 min, 4 °C.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
87. Remove the supernatant and resuspend the cells in 2 mL of ice-cold DPBS-/-.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
88. Count the cells with Trypan blue dye exclusion.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
89. Transfer 1 x 105 cells/tube for flow cytometry analysis.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
90. Keep the cell suspension in the original 15 mL conical tube and collect the pellet for DNA extraction.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
91. Spin at 100 x g, 4 min, 4 °C.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
92. Resuspend the cells (1 x 105 cells/tube) in 300 µL of ice-cold buffer for FACS.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
93. Add 5 µL of 7-AAD into a tube with the cells and incubate at Room temperature for 10 min.
 


SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
94. Culture NSC cells into a 60-mm culture dish until the cells cover the dish area, at 85-90% confluence. It is approximately 6-7 days.
 

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
95. Prior to starting the dissociation, coat the fresh 60-mm culture dishes with 3 mL/dish of cold Geltrex™ and place it into the incubator at 37 °C for at least 30 min.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
96. Remove Geltrex™, and immediately add 2 mL/dish of fresh NEM medium to prevent drying.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
97. Place fresh culture dishes into the incubator at 37 °C.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
98. Carefully aspirate the old medium from the cell culture dish.
SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
99. Wash the dish with 2 mL of DPBS-/-, remove immediately.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
100. Add 2 mL of Accutase™ into the 60-mm culture dish, ensure that cover of dish area.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
101. Incubate the cell suspension at 37 °C for 3-5 minutes.
 


SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
102. Add 3 mL of DMEM/F12 and gently triturate the clusters across the dish surface.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
103. Transfer the unattached cell suspension into a 15 mL conical tube containing 3 mL of DMEM/F12 using a 5 mL serological pipette.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
104. Spin down at 300 x g, 4 min, 25 °C.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
105. Aspirate and discard the supernatant.
SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
106. Wash with 5 mL of DMEM/F12 using a 5 mL serological pipette.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
107. Count the cells with Trypan blue dye exclusion.
SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
108. Spin down at 300 x g, 4 min, 25 °C.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
109. Resuspend the cell pellet with fresh growth NEM medium using a 5 mL serological pipette.
 

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
110. Add the cell suspension at 1 mL/dish into fresh 60-mm culture dishes previously prepared at steps 94-97. Should have a total volume of 3 mL/dish.
SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
111. Incubate the cells at 37 °C, 95% O2, 5% CO2 in humidified air.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
112. After 24 h, replace the old growth medium with 3 mL of fresh NEM medium.

SECTION: Cell expansion of the NSC cell line ● Timing 14-20 d-Passage cells ● Timing 30 min
113. Split the cells every 6 days.
SECTION: Maintenance of NSC cell culture for 5 min daily
114. Carefully aspirate the old growth medium from the cell culture dish.
SECTION: Maintenance of NSC cell culture for 5 min daily
115. Replace with 4 mL of fresh growth NEM medium.

SECTION: Maintenance of NSC cell culture for 5 min daily
116. Incubate the cells at 37 °C, 95% O2, and 5% CO2 in humidified air until they reach 80-90% confluent.

SECTION: Maintenance of NSC cell culture for 5 min daily
117. Change the growth medium every 2 days.
 

SECTION: Cells preparation for electroporation ● Timing 6 d
118. Replace the old growth NEM media for 6 days.
 

SECTION: NSC cell electroporation ● Timing 1 h
119. (Optional) Prior to starting dissociation, add iROCK (10 micromolar (µM)) into 60-mm culture cells dish for 60 min.

SECTION: NSC cell electroporation ● Timing 1 h
120. Coat the fresh 12-well plate with 6 mL of cold Geltrex™ (0.5 µL/well) as described in steps 95-97.
SECTION: NSC cell electroporation ● Timing 1 h
121. Aspirate Geltrex™ and add 0.5 µL/well of antibiotic-free fresh NEM medium with iROCK (10 micromolar (µM)).

SECTION: NSC cell electroporation ● Timing 1 h
122. Carefully aspirate the old growth medium from the cell 60-mm culture dishes.
SECTION: NSC cell electroporation ● Timing 1 h
123. Wash with 2 mL/dish of DPBS-/-, and remove immediately.

SECTION: NSC cell electroporation ● Timing 1 h
124. Add 2 mL/dish of Accutase™, ensure that cover of dish area.

SECTION: NSC cell electroporation ● Timing 1 h
125. Incubate the cell suspension at 37 °C for 3-5 minutes.
 



SECTION: NSC cell electroporation ● Timing 1 h
126. Add 2 mL/dish of DMEM/F12 and gently triturate the clusters across the dish surface.

SECTION: NSC cell electroporation ● Timing 1 h
127. Remove unattached cells using a 5 mL serological pipette.
SECTION: NSC cell electroporation ● Timing 1 h
128. Transfer the cell suspension into a 50 mL conical tube containing 10 mL of DMEM/F12.

SECTION: NSC cell electroporation ● Timing 1 h
129. Spin down at 300 x g, 4 min, 25 °C.

SECTION: NSC cell electroporation ● Timing 1 h
130. Remove the supernatant and wash with 5 mL of DPBS-/-.

SECTION: NSC cell electroporation ● Timing 1 h
131. Count the cells with Trypan blue dye exclusion.
SECTION: NSC cell electroporation ● Timing 1 h
132. Spin down at 300 x g, 4 min, 25 °C.
 


SECTION: NSC cell electroporation ● Timing 1 h
133. Resuspend the cells with ice-cold DPBS-/-.
SECTION: NSC cell electroporation ● Timing 1 h
134. Gently mix the suspension cells and transfer 2 mL/15 mL conical tube.
SECTION: NSC cell electroporation ● Timing 1 h
135. Spin down at 300 x g, 4 min, 4 °C.

SECTION: NSC cell electroporation ● Timing 1 h
136. Remove the supernatant and resuspend the cells with 1S electroporation buffer mix with the plasmids in a total volume of 100 µL.
 


SECTION: NSC cell electroporation ● Timing 1 h
137. Immediately transfer the suspension cells into the 0.2 cm electroporation cuvette, avoid bubbles.
SECTION: NSC cell electroporation ● Timing 1 h
139. Immediately add 1 mL of fresh NEM media with iROCK (10 micromolar (µM)) into the electroporation cuvette using a micropipette with a 1000 µL tip to avoid low cell viability.

SECTION: NSC cell electroporation ● Timing 1 h
140. Immediately transfer the cell suspension into a 15 mL conical tube containing 0.5 mL of antibiotic-free fresh NEM media with iROCK (10 micromolar (µM)).
 

SECTION: NSC cell electroporation ● Timing 1 h
141. Gently mix the cell suspension and transfer into three wells (0.5 mL/well) of the coated Geltrex™ 12-well plate previously prepared in step 28.
SECTION: NSC cell electroporation ● Timing 1 h
142. Incubate the cells at 37 °C, 95% O2, 5% CO2 in humidified air.

SECTION: NSC cell electroporation ● Timing 1 h
143. After 24 h, replace the old growth medium with antibiotic-free fresh NEM medium.
 

SECTION: Postelectroporation ● Timing 2 d
144. After 48 h of transfection, the integrase activity can be evaluated by using flow cytometry.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
145. Harvest cells using Accutase™.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
146. Remove the old growth medium and wash with 0.5 mL/well of DPBS-/-, remove immediately.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
147. Add 0.4 mL/well of Accutase™.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
148. Incubate the cells in the incubator at 37 °C for 5-10 minutes.
 


SECTION: Preparation of sample and flow cytometry ● Timing 2 h
149. Add 0.5 mL/well of DMEM/F12 into a 15 mL conical tube containing 5 mL of DMEM/F12.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
150. Spin down at 300 x g, 4 min, 25 °C.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
151. Wash the cells with ice-cold DPBS-/-.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
152. Spin at 300 x g, 4 min, 4 °C.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
153. Remove the supernatant and resuspend the cells in 2 mL of ice-cold DPBS-/-.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
154. Count the cells with Trypan blue dye exclusion.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
155. Transfer 1 x 105 cells/tube by flow cytometry analyses.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
156. Keep the cell suspension in the original 15 mL conical tube and collect the pellet for DNA extraction.
SECTION: Preparation of sample and flow cytometry ● Timing 2 h
157. Spin at 300 x g, 4 min, 4 °C.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
158. Resuspend the cells (1 x 105 cells/tube) in 300 µL of ice-cold buffer for FACS.

SECTION: Preparation of sample and flow cytometry ● Timing 2 h
159. Add 5 µL of 7-AAD into a tube with the cells and incubate at Room temperature for 10 min.
 

SECTION: PBMC isolation ● Timing 1.5 h
3. With a 25 mL serological pipette, add ~35 mL of blood at the top of the 10 mL tube containing Ficoll-Paque at Room temperature.
 

SECTION: NSC cell electroporation ● Timing 1 h
138. Electroporate the cells by using the Nucleofector 2D program A-33.