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SOP | Marion Frankenberger

30-Apr-2011

SOP: Sputum sample processing

Date: Experimenter: Experiment description: Author: Marion Frankenberger Date: 30/04/2011 Version: 1.0

Please note that sputum is to be processed immediately and without delay. List of materials 1. 2. 3. 4. 5. Sputolysin, Calbiochem #560000 (dilute provided stock solution 1:10 with aqua dest.) alternatively DDT as a 1% (w/v) monthly stock can be used, dilute 1:10 on the day Bench Spiromix end-over-end , to hold different size tubes 2 ml Eppendorf tubes, 15 and 50 ml polypropylene tubes BD Falcon cell strainer 100 µm and 40 µm (#352360 and #352340) or 48 µm gauze 15 ml v-bottom polypropylene tubes #352095 (BD Falcon) Trypan blue 0.4%, Sigma #T8154 Neubauer counting chamber Shandon cytocentrifuge Polylysine microscope slides (# MPR-455-L, Fisher) Plastic spatula (Brand, 97877 Wertheim,Germany), blue tip pipette 2 ml Sarstedt tubes #72.694.416

Weigh an empty 15 or 50 ml tube transfer the entire volume of sputum (avoid saliva)°1 reweigh and record the sample weight based on the weight in g add 2 times the volume in ml of sputolysin (Calbiochem #560000) disperse sputum by repeated gentle aspiration into a plastic Pasteur pipette, vortex for 15 seconds and incubate at RT for 15 min on a bench Spiromix°2 6. add PBS, same volume as sputolysin, vortex for 15 sec 7. sieve through cell strainer, (first 100 µm, followed by 40 µm) into a polypropylene tube of appropriate size, record the volume°3 spin suspension 10 min 800 g at RT. 8. harvest supernatant, transfer it in aliquots of 0.5 - 1 ml into 2ml Sarstedt tubes #72.694.416 and store them at -20ºC. 9. resuspend cell pellet in 0.5 - 1 ml of PBS °4 10. determine cell number and composition * Mix 10l of cell suspension with 10l of Trypan blue [dilution factor = 2] or 90l of Trypan blue [dilution factor = 10] * Fill a Neubauer chamber and count viable leukocytes, dead leukocytes and squamous cells (whether viable or not). (See example in figure). Cells touching the top and left lines are counted, cells touching lower and right lines are not.

Squamous Spore

Leukocyte

SOP | Marion Frankenberger

30-Apr-11

Calculate the number of viable leukocytes / ml, the percentage of dead leukocytes and the percentage of squameous cells a) number of cells in four 16 field squares (red below) divided by 4. This is multiplied dilution factor (2 or 10) = cells x 104 /ml

b)

dead leukocytes x 100 % dead leukocytes = [viable + dead leukocytes]

c)

% squamous cells

=

Squamous cells x 100 [Squamous cells + viable leukocytes + dead leukocytes]

(This is the only calculation involving squamous cells) Calculate the total number of cells (cells/ml x total ml) Calculate cell count per g sputum = total number of cells / total weight (g) of sputum Enter results into the cell number sheet (last page) When you have less than 1 x 105 cells stop here and record this as failure and discard the supernatant 11. When you have 1- 2 x 105 cells go ahead and prepare 2 slides as given under 11. and stop here When you have > 3 x 105 cells proceed until the end Prepare 2 slides with about 1 x 105 cells each ( in 100 or 200 µl depending on your centrifuge) in a cytocentrifuge (18 g = around 400 rpm, for 6 minutes) 12. Use the rest of the cells for further experiments like mRNA expression or FACS analysis

SOP | Marion Frankenberger

30-Apr-11

Footnotes °1 °2 °3 °4 picking plugs is done in many laboratories but in our experience gives 10 times less cells not 37°C incubation since this may lead to ex-vivo release of mediators gauze can be used for the larger volumes, take care to avoid fluid loss before centrifugation samples may be too dilute for appropriate counting

Flow cytometry analysis of sputum macrophage subsets For determination of small and large sputum macrophages before and after RosetteSep isolation the following monoclonal antibodies according to the manufacturer's instructions are used: anti-CD66bFITC (#0531, Immunotech, Coulter, Krefeld, Germany), anti-CD16b-PE (#550868, Pharmingen, BD Sciences, Heidelberg, Germany), anti-CD14-PC5 (#A07765, Coulter, Krefeld, Germany). Small and large macrophages are determined in percent of all macrophages by first excluding the CD66b/CD16b-positive granulocytes and further gating on the CD14-positive macrophages. Within this population small and large macrophages are re-gated in a forward versus side scatter plot to define their percentage and purity.

Sputum Cell Number Sheet

Donor-Code

Weight of Sputum

Weight of empty centrifuge tube (g) Weight of tube + selected sputum (g) Weight of selected sputum (g) =W Vol of Sputolysin added: (2 x W) (mL)

Volume of PBS added (mL) = Vol of Sputolysin

Haemocytometer Counts (mean of four counted 16 field squares)

Leukocytes % (viable+dead) Squamous Viable cells for Leukocytes Non-viable cells for Leukocytes % Dead leukocytes

Squamous

Calculation of Total Number of Cells in Selected Sputum Sample

Weight of Selected Sputum `W' Resuspension volume used Dilution used Mean number of viable cells in one square 16 field of haemocytometer Total viable cells x 106 in sample Total Cell Count per gram

g

ml

x 106

x 106/g

Signed:............................................................Date:

Day Month Year

Version 1.1 / Apr-11

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