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Descripción del Protocolo

NotaPasos

• Immortalized human keratinocytes (HaCat) were cultured in DMEM/Hams F12 medium supplemented with bicarbonate, an antibiotic-antimycotic, and 10% FBS (GIBCO), at pH 7.2. Cells were sown in treated culture plates (75 cm2) (Nest), and cultured at 37 °C in a 5% CO2 . Medium was changed every third day. When the keratinocytes were 80% confluent in the culture plate, they were harvested for the assays. Cell harvest was done by extracting medium with a sterile pipette, washing with PBS (pH 7.2), extracting the buffer and adding 3 ml 0.05% trypsin EDTA. The plate was incubated at 37 °C for 4 mins to activate the enzyme and disrupt cell-cell and cell-matrix interactions. Cell detachment was confirmed with an inverted microscope. Because FBS inhibits trypsin-EDTA and therefore its activity, 5 ml medium was added to the box. The total volume in the plate was recovered and placed in a 15 ml tube (Nest). After centrifuging at 1500 rpm for 5 mins, the supernatant was removed and 1 ml medium added. The tube was mixed gently to disperse the cells. Cell counts were done using 10 µl of this mixture in a manual hemocytometer and expressed as cells in 1 ml. Based on the cell counts, calculations were done of the amount of medium (ml) required per ml cell-containing medium, considering the number of cells to be sown in each well and well volume (ml).

• For the assay, a keratinocyte density of 5 x 104 cells in 300 µl medium per well was used . This was placed in each well of treated 48-well plates (Nest), the plate was incubated and growth monitored until 80% confluence was attained. This was achieved 24 h after sowing, at which time the wound was made in the cells. The culture medium was removed from each well and two washes done using 300 µl fresh, sterile PBS 1X 7.2. During the second wash, a scratch wound was made in the cells using a 1 ml pipette tip (blue) by applying enough force to disrupt the cells without damaging the plastic. The PBS 1X pH 7.2 was removed and a third wash done to remove as much debris as possible, after which PBS was placed over the cells.

• Quantification of cell wound healing in the presence of the peptide fractions was done by adding them to the wounded cells and monitoring the healing process. A negative control (medium alone) and positive control (10 ng/mL EGF; GIBCO) were used. Fractions ( hidrolized collagen minor of 3 kDa and peak 2 ) were dissolved in culture medium (12% DMEM/HAMS) at a 3.5 mg/ml concentration. The PBS in the well was removed, and the fractions and controls added to the wells. Six replicates were done per control and per fraction. The plates were incubated at 37 °C and 5% CO2. The healing process was monitored at 24 after wound induction (time zero, 0 h), and the experiment stopped when any of the controls or fractions exhibited 90% healing. Images were taken at 0 and 24 by placing a plate under an inverted, phase-contrast microscope (10X) equipped with a digital camera. A positive control was added into the in vitro assay ,rh-fgf-b (thermo Fisher) as well as control without wound and a control of wound with only media.

• Using the images, healing rate was measured using the Tscratch software (Gebäck, Zurich, Switzerland).14 Images were analyzed per condition, per time point, and means and standard deviations calculated. A Student’s t-test was applied to analyze the results; significance was set at P<0.05.

Notas

• Pico 2 menor de 3 kDa Se pesaron 3.5 mg/ml (mililitros de medio) Al final en 300\(\mu\)l debe haber una concentración de 0.2 mg /ml

V1C1=V2C2 V1= [No pozos x 300][0.2 mg/ml (C2)] 3.5 mg /ml (C1)

V2 = (Número de pozos x 300\(\mu\)l medio)

• Condiciones
  • Control sin herida
  • Control con herida y solo medio
  • Control + Factor crecimiento (3\(\mu\)l/ml de medio) rh-fgf-b thermo Fisher. Puesto que no teníamos otro factor
  • Muestras: pico 2 proveniente de col hidrolizado menor de 3 kDa, colágeno hidrolizado menor de 3 kda

Diagrama de Flujo

Figura

Anexos

Material

• Mouse Macrophage Nucleofector Kit
  (Lonza, Cat# VPA-1009, 25 Reactions)
• Mission esiRNA Mouse F11r
  (Sigma, Cat# EMU082531-50UG, 200 ng/µL, 250 uL)
• siRNA Universal Negative control #1
  (Sigma, Cat#SIC001-5x1NMOL, 1nmole/tube, MW 13,317 g/mole)
  dissolve in 66.6 µL H2O to make 200 ng/µL solution.

Documentos de ?@sec-ref

(cite?) 10.1002/9780470089941.et0702s10

actualizaciones

versión		Elaboró	Revisó
2023.04	inicial	Oscar Medina Contreras	Oscar Medina Contreras
2024.01	actualización	Oscar Medina Contreras	Oscar Medina Contreras

siRNA

Macrophage
 siRNA  protocol Feb 12, 2012 Wooki Kim

1. Prepare 12-well plates by filling appropriate number of wells with 1.5 ml culture medium (RPMI+10% heat inactivated FBS+penicillin/streptomycin) and pre-incubate/equilibrate plates in a humidified 37°C/5% CO2 incubator
2. Harvest bone marrow cells from either WT or JAM-A KO mice in PBS and determine cell concentration.
3. 4. Adjust cell concentration at 1 x 106 cell/mL Centrifuge 1 mL of cell suspension (1 x 106 cells per sample) at 200x g for 5 minutes at room temperature.
4. Discard supernatant completely.
5. Resuspend the cell pellet carefully in 100 μl room temperature Nucleofector® Solution per sample.
6. Avoid storing the cell suspension longer than 15 minutes in Mouse Macrophage Nucleofector® Solution, as this reduces cell viability and gene transfer efficiency
7. Transfer cells into a provided cuvette (sample must cover the bottom of the cuvette without air bubbles).
8. Input 10 µL of siRNA (or scrambled control) directly into the cuvette.
9. Close the cuvette with the cap
10. Select the Nucleofector® Program Y-001 (Whitehead 455C)
11. Insert the cuvette with cell/DNA suspension into the Nucleofector® Cuvette Holder and apply the selected program
12. Take the cuvette out of the holder once the program is finished (transfection occurs in a blink!).
13. Add 400 μl of the pre-equilibrated culture medium to the cuvette and gently transfer the sample into the 12-well plate (final volume of 2 ml media per well). Use the supplied pipettes and avoid repeated aspiration of the sample
14. incubate the cells in humidified 37°C/5% CO2 incubator for 8 hr.
15. Aliquot 150 µL cell suspension into round bottomed 96-well plates, and add 50 µL zymosan suspension (40 µg/mL).
16. Incubate additional 20 hr and harvest supernatant by centrifuge.
17. Determine cytokine/chemokine production by ELISA kits.