''First PART'': Isolation and plating of Monocytes, Lymphocytes, Neutrophils and Platelets from human blood without activation.
''Second PART'': Measurement of mitochondrial function for monitoring pathological processes and the impact of treatments. ^^key words (monitoring reactive oxygen species generation and bioenergetics in a clinical setting)^^.

''?'' Collect blood usual ''2'' tubes (minim ''8 mL'' max 20mL) in BD Vacutainer ACD tubes (with yellow caps) ^^Cat. No # REF 364606^^ 8.5 mL 16x100mm) ^^ contain 1.5 mL of ACD (Anticoagulant Acid Citrate Dextrose)^^.
After drawing, the blood should be allowed to cool at Room Temperature for minimum 30 - 45 minutes. If used immediately after being drawn, the number of Mononuclear cells collected will be low. Separation procedures are optimal when both blood and Histopaque (Ficoll) are at 18 – 20 °C, with an acceptable temperature range of 18 – 26 °C.

''?'' Centrifuge whole blood at ''1900 rpm'' (rcf 500 × g) for ''15 min'' in a Centra CL 2 centrifuge (Rotor IEC 236 - swinging bucket rotor).

''?'' Remove the top layer that contains the Platelet Rich Plasma (''PRP'') ^^plasma riche en plaquettes 55% of blood^^ with a transfer pipette until ''1 cm'' remains above the cell layer (Erythrocyte/Buffy Coat). Set aside the PRP at room temperature for processing later at step ?. ^^High lipid levels, rheumatoid factor, anemia, and drug treatment are all possible causes for poor separation of a specific donor’s blood. If the plasma is not clear, this is an indication of high lipid levels.^^

''?'' Transfer the ''Buffy Coat'' ^^1% of blood^^ [aprox. 4 - (6) mL] 1 +1 cm to a sterile conical Tube50mL and dilute to 24 mL with basal RPMI to at least 4x the starting buffy coat volume (1:4). Mix gently. ''RPMI 1640 (Wisent Inc) Cat # 350 - 046 CL (no antibiotics, no phenol red, no FBS) contain 4 mM L-Glutamine at 37 ºC''.

''?'' Prepare the Density Gradient. Double gradient is formed by layering an equal volume of 1119 and 1077: Take out the Ficoll from frigo one day before. Add first 3 mL of low density ''Ficoll 1077'' (with a specific gravity of 1.077) to each tube in three conical ''Tubes15mL'' / 8 mL blood.

^^''Details:'' (''1'') Histopaque® - 1077 (sterile-filtered, density: 1.077 g / mL) from Sigma, Cat. No #10771 for 100 mL; (''2'') Histopaque®-1119 (sterile-filtered, density: 1.119 g / mL) Cat. No # 11191 for 100mL. Temperature is extremely important when performing the procedure. A 100 mL bottle of Histopaque (Ficoll) stored at 2 – 8 °C may take several hours to reach 18 – 20 °C. When planning to use, we recommend removing the Histopaque from the refrigerator the previous day and let the bottles stand on the bench overnight. This ensures the solution is at room temperature and ready for use. When using both 1077 and 1119 to isolate neutrophils, careful technique must be used to prevent mixing at the solution interface. Check the integrity of the interface using Schlieren Optics. Any swirling or mixing at the interface between the layers should be evident when holding the tube up against the light. If the interface was properly prepared, there should be a sharply demarcated line. If the sharply defined line is not present or if swirling is present at the interface, discard the tube and start over. It is also important to use the gradient as soon as it is formed. There are no chemical differences between 1077 and 1119; the two solutions have the same components and ''will start diffusing together over time''. When this happens, recoveries will be poor or nonexistent.^^

''?'' In the morning by placing the pipette tip beneath (below) of 1077 add slowly 3 mL of high density ''Ficoll 1119'' without mixing with the upper gradient (3 mL / minute). Use Syringue pump Model Sage Instrument Model 355 (Syringue 60 mL diameter ? = 26.59mm RANGE: off - X¹/10 and %FLOW: 500).

''?'' A total of 6 mL of density gradient media should be present at this stage with a visible phase of separation at the 3 mL mark. In the tube will be: Ficoll low1077 layer up and Ficoll high1119 layer down.

''?'' Add on the surface of gradient, ''gently'', ''8 mL'' of diluted blood (from step ?) to each gradient tube using the low-power setting to prevent disturbing the gradient layers. (//better way with SyringuePipette//) The total volume should be 14 mL at this step. Centrifuge tubes at ''2200 rpm'' (rcf 700 x g) for ''30 min'' at room temperature.

''?'' Three distinct cell bands should be evident. The upper most bands (between 1077 and plasma) contains ''M''ononuclear cells (''M''NCs) and Platelets, and the middle band (between 1007 and 1119) contains ''P''olymorphonuclear (''N''eutrophiles) cells (PM''N''s) and lower band (below 1119) contains RBCs.

''?'' Detail of three distinct cell layers (see details on graph) click here blood layers.

''?'' Collect the ''M''NC by using sterile ''glass pipettes'' without disturbing the other cell bands. Combine the ''M'' population from each tube into a sterile conical Tube50mL.
Add ''4'' volumes of RPMI to the Tube50mL containing the MNC fractions respectively to dilute the density gradient.

''?'' Repeat this for the PM''N'' population as well and add ''10'' volumes of RPMI to the ''N'' fractions to dilute the density gradient.

''?'' & ''?'' Centrifuge tubes at ''2200 rpm'' (rcf 700 × g) for ''10 min'' at room temperature. Discard the supernatant.

''?'' & ''?'' Resuspend ''M''NC cell pellet and PM''N'' cell pellet in 1 mL RPMI buffer containing 0.5% ultra-pure fatty acid-free BSA (Milteny-BSA) and transfer to sterile tube ''Eppendorf''. ^^NOTE dilution^^//(Milteny-BSA # 130-091-376 add 2500 µL BSA in 50 mL RPMI)//. ''Pellet'' (spin down) the cells using a benchtop picofuge for ''30 sec''. Discard the supernatant. Resuspend each cell pellet in ''80 µL'' RPMI buffer-BSA.

^^NOTE centrifugation^^ FOR Tube Eppendorf - Pellet (spin down) the cells using a benchtop picofuge for 30 sec. FOR Tubes15mL - Centrifuge at 1400 rpm (rcf 300 × g) for 5 minutes.

''?'' Add ''20 µL'' of magnetic beads (from the frigo at 4 ºC) labeled-antiCD''14'' antibody to the tube containing the ''M''NC fraction for positive selection of ''M''onocytes. For the tube containing the PM''N'' cell fraction, add ''20 µL'' of magnetic beads labeled-antiCD''15'' antibody for positive selection of ''N''eutrophils. ''Mnemonics: M14 and N15''

''?'' Mix well with tips and no bubbles, and incubate for 15 min at 4 ºC in the frigo.

''?'' Wash each cell suspension with 1 mL RPMI-BSA. ''Pellet'' (spin down) the cells using a benchtop picofuge for ''30 sec''.

''?'' Discard the supernatant. Resuspend each pellet in ''500 µL'' RPMI-BSA.

''?'' Proceed to magnetic separation.

''?'' Place column in the magnetic field. Prepare (activate) LS column (one for each cell suspension) by rinsing with 3 mL buffer MACS PBS - BSA.

''?'' Apply ''500 µL'' cell suspension onto the column. Attention: ''Collect unlabeled cells which pass through'' that means collect total efluent. ''This is ''M''(-) the unlabeled CD14 (-) cell fraction containing ''L'' cells''. Wash column with 3 mL buffer. Perform washing steps by adding buffer three times, each time once the column reservoir is empty. (3mL + 3mL + 3mL + 500µL).

''?'' To isolate monocytes and neutrophils. Remove column from the separator and place it on a suitable collection tube. Pipette 5 mL of buffer onto the column. Immediately fush out fraction (+) with the magnetically labeled cells by firmly applying the plunger supplied with the column. This is the labeled CD14 (+) monocytes ''M'' and respectively CD15 (+) neutrophils ''N'' cell fraction. (Ready for counting or reduce the volume to 1 mL for count).

''?'' To isolate lymphocytes ''L''. Centrifuge ''1400 rpm'' (rcf 300 × g) for ''10 min'' the flow-through-wash fraction of the ''M''NCs (-) from step ?. Discard the supernatant and resuspend the cell pellet in ''80 µL'' buffer. Add ''20 µL'' of CD61 and ''20 µL'' CD235a antibodies in the same Eppendorf.

''?'' Mix well and incubate cell suspension for 15 min at 4 ºC.

''?'' Repeat the MACS separation as before ? - ? and collect ''the flow'' (negative selection) though containing the ''L''ymphocytes ''L''. This is the labeled CD61(-) and CD235a (-) lymphocytes fraction. (Ready for counting or reduce the volume to 1 mL for count).

''?'' In the column are the cells magnetic labeled CD61(+) megakaryocytes ^^responsible for the production of blood thrombocytes (platelets)^^ and also platelets (plaquettes) and CD235a (+) erythrocytes or //(Érythrocytes)// or //(RBC)//. Discard.

''?'' To pellet ''M''onocytes, ''N''eutrophils, and ''L''ymphocytes fractions: Centrifuge tubes at ''2200 rpm'' (rcf 700 × g) for ''10 min'' at room temperature. Discard supernatants. Cell pellets (//les culots//) should be resuspended in ''1 mL'' extracellular flux media (XF-DMEM) for counting.

''?'' ''8 mL'' of whole blood should result in 1 - 5 × 10^^6^^ ''M''onocytes / mL, and 5 - 20 × 10^^6^^ ''L''ymphocytes and ''N''eutrophils / mL.

''?'' To isolate ''P''latelets: Centrifuge ''P''RP tubes (from step ?) ''3200 rpm'' (rcf 1,500 × g) for ''10 min'' at room temperature. Remove the plasma. Wash cell pellet once with sterile ''5 mL'' buffer (PBS Macs +BSA) supplemented with ''1'' µg/mL ''PGI2'', ^^PGI2 Prostacyclin (also called Prostaglandin I2) {//Sir John Vane + Salvador Moncada, R Gryglewski S Bunting//} is a lipid - inhibits platelet activation. Cat. No: # 18220, Cayman Chemical^^. Centrifuge ''3200 rpm'' (rcf 1,500 × g) for ''10 min'' at room temperature. Suspend final pellet in ''1 mL'' of PBS - PGI2 buffer.

Determine platelet count by turbidimetry once the platelets are suspended in PBS-PGI2 buffer by microplate reader as described by Walkowiak et al. 1997 (PMID 9253804) using the following equation: [6.23/(2.016 – (1.33×750×?/750) – 3.09 ]× dilution factor = # x 10^^8^^ platelets/mL/. In plate 98wells the meniscus works as an optical lens and refract the optical path extinction (?) value was corrected by substraction of reference sample exctinction. ? = 0.5842666 (OD) at 750 nm.

!Preparation of ''PGI2'' 1 mM (synonym Prostacyclin) Prostacyclin (sodium salt, Cat No: #18220, Cayman Chemical) FW: 374.5 is unstable at neutral or acidic pH soluble in (50 mM Tris-HCl pH 8.87). (SS) [''1 mM''] Stock Solution: 1mg add in 2670 µL Tris 50 mM and divided in 20?µL aliquots in eppendorf [store at -20°C or - 80°C for weekly use]. (SF) [''0.5 µM''] Final Solution: ''3'' µL in 6 mL Buffer-BSA. ^^PGI2 is used at each step of platelet washing procedure at a final concentration of 0.5 µM (0.5 µL of the stock solution for 1 mL of platelet suspension. Since the half-life of PGI is short (a few minutes), it must be added to the washing solution just before centrifugation or platelet resuspension. The PGI solution should be stored at 4°C immediately after thawing and should not be frozen again^^

!Preparation of Tris 50 mM. FW:121.14 (SS) Tris [50 mM] for 1L solution first weigh out 6.057 g Tris in water; Every 1 g of Tris requires 165 mL of water to be added. (SF) [''50 mM''] Tris : 908.55 mg add in 150 mL water add HCl 01M for pH 8.87.

!''Plating of the Cells - CellTak Method'' See the tiddly Design Plate 96 wells and [[ [0] Platform synchron]]. Procedure for CELTAK PLATE PREPARING //Dispence within 10 minutes// (for 69 wells): add 210 µL Cell-Tak to 420 µL dH~~2~~O, 1453 µL of 0.1N Sodium Bicarbonate and ±105 µL of 1N NaOH to pH 8,0 - 8,5. Place ''30'' µL diluted Cell-Tak into each well. Shake delicate. Allow at least ''20'' minutes for adsorption. Flick or aspirate off the Cell-Tak solution and wash with distiled water, air dry before storing at 2 - 8 °C for approximately two weeks. Adhesive extracted from //Mytilus edulis//. An aid to attaining this pH window is to use a volume of NaOH equal of half the volume Cell-Tak solutions. Put Cell-Tak also in the Blank Control of SeaHorse plate.

|cssClass|k |!cel tak ~~B~~|!H~~2~~O|!Bicarb|!NaOH|!Total|!wells30µL|h | 41 | 400 | 820 | 20.5 | 1281 | 41 | | 80 | 150 | 540 | //± 40// | 770 | 25 | | 210 | 420 | 1453 | //± 105// | 2083 | 69 ? | |z | z |z |//± z// | z |64 × 2 ? | |307 |z |z |//± z// |z |96 × 1=96| |614 |z |z |//± z// |z |96 × 2=192| |1229 |z |z |//± z// |z |96 × 4=384|

new formula pH 8,0 - 8,5.

!Seeding
250k cells/well for ''M''onocytes, 250k cells/well for ''L''ymphocytes, 250k cells/well for ''N''eutrophils, 25 x 10^^6^^/well for ''P''latelets. Alternatively, to measure oxidative burst response, Neutrophils can be seeded at 125 k cells/well. The final seeding volume for each cell suspension should be 200 µL/well. For platform synchron the cells should be seeded in microplate ~150 000 cells/well. Centrifuge the plate at ''200 rcf'' for ''30 sec'', rotate the plate 180° and centrifuge again at ''300 rcf'' for ''30 sec''. Centrifuge Eppendorf 5430R, with rotor A-2-MTP Cat.No: # 5430/5430 START / STOP / OPEN. //Bring final well volume to ''660 µL'' with XF-DMEM and incubate at 37 ºC for 30 min prior to XF assay//.

!NOTES ? RPMI no phenol red [1] RPMI Cat # 17 - 105 - CV (no antibiotics, no phenol red, no FBS) ? 0 mM L-Glutamine. [2] RPMI 1640 (Wisent Inc) Cat # 350 - 045 - CL(no antibiotics, no phenol red, no FBS) ? 0 mM L-Glutamine (add 2 mM). [3] RPMI 1640 (Wisent Inc) Cat # 350 - 046 - CL(no antibiotics, no phenol red, no FBS) ? with 4 mM L-Glutamine. [4] DMEM XF buffer ? 4 mM L-Glutamine, glucose and pyruvate.

? ? orthography //pellets// ? //platelets// ? In plate 98wells the meniscus works as an optical lens and refract the optical path extinction (?) value was corrected by subtraction of reference sample extinction. ? = 0.5842666 (OD) on Excell turbidity formula [ 6.23/ (2.016 - ( 1.33 × 750 × 0.5842666/750 ) ) - 3.09 ] ×1= 1.9385518 [ 6,23/ (2,016 - ( 1,33 × 750 × 0,5842666/750 ) ) - 3,09 ] ×1=1,9385518 or on Casio Scientific Calculator (29355?+4)/100(-95? + 144) × dilution factor = # 10^^8^^ platelets/mL/^^ ? https://en.wikipedia.org/wiki/Plasmapheresis ? Histopaque is a density gradient cell separation medium of Ficoll and sodium diatrizoate adjusted to a density of x g / mL Diatrizoate is a radiocontrast agent, containing iodine.is considered a high-osmolality contrast agent. Its osmolality ranges from approximately 1500 mOsm/kg (50% solution) to over 2000 mOsm/kg (76% solution). ? Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets (Victor Usmar). On google keyword: Jove e51301 oxidative burst (52201). For video: Ctr+Shift+K web console "Net" and "Logging" check video extension ? Detail for download open video flash 1. open jove page 2. open Chrome Inspector by pressing these 3 keyboard buttons together: Ctrl Shift i and (Ctr+Shift+K for Firefox) 3. Click the tab marked "Network" at the top of the Inspector window 4. refresh the webpage from the Inspector by pressing these keyboard buttons together: Ctrl R 5. shortly after you hear the audio playing in the background, you should see a line in the Inspector for the video 6. Identify the video by looking at the 'Type' column, find the only entry for 'video/mp4' or 'video/quicktime' 7. right-click on the video entry's first column (column name 'Path'), then click 'copy link address'

? Counting Chamber Hausser Scientific Company (Fuchs-Rosenthal ) #Cat.No.: 3720. Mix 10 µL cells + 10 µL (0.4%) Trypan Blue stain Invitrogen Gibco #Cat.No.: 15250-061 (storage 15-30°C) in counting chamber 20 µL. Count 16 squares (1 mm^^2^^).

¦ ? ? ? ? ¦ = 24 ¦ ? ? ? ? ¦ = 29 ¦ ? ? ? ? ¦ = 26 ¦ ? ? ? ? ¦ = 25 ========== = 104 cells in 16 ? 104 × dilution 2 × 5000 = 1 040 000 cells/ mL = 1.04 M 1.04 M × 5 mL total = 5.2 Mil. cells total.

? Counting Bright-Line™ Hemacytometer Sigma #Cat.No.: Z359629. Mix 10 µL Blue + 10 µL cells in counting chamber and count 5 squares (from 25) big 25 of 25. ================ ¦? | ? | ? | ? | ?¦ = 52 ¦? | ? | ? | ? | ?¦ ¦? | ? | ? | ? | ?¦ ¦? | ? | ? | ? | ?¦ ¦? | ? | ? | ? | ?¦ ================ = 52 cells in 5 ? = 0.52 M / mL dilution 1/2 = × dilution 2 = 1.04 M cells / mL 1.04 M × 5 mL total = 5.2 Mil. cells total

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? Convert to OD600: The alternative way to measure cellular density is by using a spectrophotometer, which gives a reading in units of OD600. An OD600 of 1 = 1 × 10^^7^^ cells / mL.

? Important conversions: (cm^^3^^ = mL) (mm^^3^^= µL) (50 µL ? 1 Drop) (250000 µL ? Cup)

? Dozaj Cristal Violet 1- Retirer le milieu de culture par aspiration. 2- Ajouter 150 µL de p-formaldéhyde-PBS (4%). 3- Incuber 5 minutes à température pièce. 4- Enlever le p-formaldéhyde par aspiration. 5- Ajouter 150 µL de cristal violet (0,1%-H2O) et incuber 5 minutes à température pièce. 6- Enlever le cristal violet par aspiration. 8- Faire 2 lavages avec 1000 µL de PBS. 9- Ajouter 150 µL de PBS.. 11- Lire la D.O. à 595 nm. (reference 0 nm) (p-formaldéhyde 4% est fait en dissolvant 4 g de p-formaldéhyde dans 100 mL de PBS sous barreau magnétique. Il peut être nécessaire de chauffer a µOnde).

? Tecan http://www.tecan.com/platform/apps/product/index.asp?MenuID=5167&ID=12045&Menu=1&Item=21.2.10.9 key words: ? Accuracy, Precision, Reliable, Reproducible, Fast, Compact, Sensitive?